Anti-c5 antibody combinations and uses thereof

ABSTRACT

The present invention relates to combinations of anti-C5 antibodies and antigen-binding fragments which have been determined to exhibit superior activity relative to that of a single anti-C5 antibody or fragment. The combinations include anti-C5 antibodies and antigen-binding fragments which do not compete with one another from C5 binding. Bispecific antibodies comprising antigen-binding domains which do not compete and/or bind the same epitope on C5 are also provided. Compositions and therapeutic methods relating to such anti-C5 combinations and bispecific antibodies are provided herein.

This application claims the benefit of U.S. Provisional PatentApplication No. 62/598,023 filed Dec. 13, 2017, which is hereinincorporated by reference in its entirety.

FIELD

The present invention is related to combinations including antibodiesand antigen-binding fragments of antibodies that specifically bind tocomplement factor C5, and methods of use thereof.

BACKGROUND

The complement system is a group of plasma proteins that when activatedlead to target cell lysis and facilitate phagocytosis throughopsonization. Complement is activated through a series of proteolyticsteps by three major pathways: the classical pathway, which is typicallyactivated by immune-complexes, the alternative pathway that can beinduced by unprotected cell surfaces, and the mannose binding lectinpathway. All three pathways of complement cascade converge onproteolytic cleavage of complement component 5 (C5) protein. Cleavage ofcomplement component 5 (C5) results in the production of fragments C5aand C5b, a process that is critical during the activation of thecomplement cascade. C5a can generate pleiotropic physiological responsesthrough binding to its receptors (Monk et al. 2007, Br. J. Pharmacol.152: 429-448). C5a is a potent pro-inflammatory mediator that induceschemotactic migration, enhances cell adhesion, stimulates the oxidativeburst, and induces the release of various inflammatory mediators such ashistamine or cytokines. C5b mediates the formation of themembrane-attack complex (MAC, or C5b-9) leading to cell lysis in thelate phases of the complement dependent cytotoxicity (CDC). Further, innucleated cells that are resistant to cytolysis by C5b-9, sublyticquantities of C5b-9 can cause cellular activation which results in cellproliferation, generation of proinflammatory mediators and production ofextracellular matrix.

Monoclonal antibodies to C5 are known in the art and have beendescribed, for example, in US Patent/Publication Nos. 9206251, 9107861,9079949, 9051365, 8999340, 8883158, 8241628, 7999081, 7432356, 7361339,7279158, 6534058, 6355245, 6074642, 20150299305, 20160051673,20160031975, 20150158936, 20140056888, 20130022615, 20120308559, and inWO2017218515, WO2015198243, WO2015134894, WO2015120130, EP2563813B1,EP2328616B1, and EP2061810B1.

Anti-C5 antibodies with high affinity and biological activity are known;however, an improvement in biological activity may lead to more potenttherapies for subjects suffering from C5-associated diseases anddisorders.

SUMMARY

Particular combinations of anti-C5 antibodies and antigen-bindingfragments exhibiting surprising and unexpected levels of biologicalactivity (e.g., reduction of red blood cell (RBC) lysis) have beenidentified. Combinations of anti-C5 antibodies and fragments which donot compete with one another for C5-binding lead to a reduction in RBClysis beyond reduction associated with a single anti-C5 antibody orfragment. Compositions and therapeutic methods relating to such anti-C5combinations are provided herein.

The present invention provides a combination (e.g., a kit) comprising afirst antigen-binding protein (e.g., antibody or antigen-bindingfragment thereof) that binds specifically to C5 (e.g., human C5); andone or more further antigen-binding proteins (e.g., polypeptides (e.g.,coversin) or antibodies (e.g., eculizumab) or antigen-binding fragmentsthereof) that (i) specifically bind to C5 at an epitope which isdifferent from that of the antigen-binding protein; and/or (ii) do notcompete with the first antigen-binding protein for binding to C5. Thefirst antigen-binding protein and the further antigen-binding proteincan be co-formulated into a single pharmaceutical formulation (e.g.,with a pharmaceutically acceptable carrier) or formulated into separateformulations (e.g., each with a pharmaceutically acceptable carrier). Inan embodiment of the invention, the first antigen-binding protein and/orthe one or more further antigen-binding protein thereof are in apre-filled injection device (e.g., pre-filled syringe or pre-filledautoinjector) or vessel. For example, in an embodiment of the invention,the first antigen-binding protein (e.g., antibody) comprises CDR-H1,CDR-H2 and CDR-H3 of a heavy chain variable region that comprise theamino acid sequence set forth in SEQ ID NO: 19, and CDR-L1, CDR-L2 andCDR-L3 of a light chain variable region that comprise the amino acidsequence set forth in SEQ ID NO: 27. In an embodiment of the invention,the further antigen-binding protein (e.g., antibody or antigen-bindingfragment) comprises (i) CDR-H1, CDR-H2 and CDR-H3 of a heavy chainvariable region that comprise the amino acid sequence set forth in SEQID NO: 3, and CDR-L1, CDR-L2 and CDR-L3 of a light chain variable regionthat comprise the amino acid sequence set forth in SEQ ID NO: 11; (ii)CDR-H1, CDR-H2 and CDR-H3 of a heavy chain variable region that comprisethe amino acid sequence set forth in SEQ ID NO: 35, CDR-L1, CDR-L2 andCDR-L3 of a light chain variable region that comprise the amino acidsequence set forth in SEQ ID NO: 43; (iii) CDR-H1, CDR-H2 and CDR-H3 ofa heavy chain variable region that comprise the amino acid sequence setforth in SEQ ID NO: 51, CDR-L1, CDR-L2 and CDR-L3 of a light chainvariable region that comprise the amino acid sequence set forth in SEQID NO: 59; (iv) CDR-H1, CDR-H2 and CDR-H3 of a heavy chain variableregion that comprise the amino acid sequence set forth in SEQ ID NO: 67,and CDR-L1, CDR-L2 and CDR-L3 of a light chain variable region thatcomprise the amino acid sequence set forth in SEQ ID NO: 75; (v) CDR-H1,CDR-H2 and CDR-H3 of a heavy chain variable region that comprise theamino acid sequence set forth in SEQ ID NO: 87, and CDR-L1, CDR-L2 andCDR-L3 of a light chain variable region that comprise the amino acidsequence set forth in SEQ ID NO: 95; and/or (vi) CDR-H1, CDR-H2 andCDR-H3 of a heavy chain variable region that comprise the amino acidsequence set forth in SEQ ID NO: 103, and CDR-L1, CDR-L2 and CDR-L3 of alight chain variable region that comprise the amino acid sequence setforth in SEQ ID NO: 95. In an embodiment of the invention, the firstantigen-binding protein (e.g., antibody or fragment) comprises a heavychain variable region that comprises: a CDR-H1 that comprises the aminoacid sequence set forth in SEQ ID NO: 21, a CDR-H2 that comprises theamino acid sequence set forth in SEQ ID NO: 23, and a CDR-H3 thatcomprises the amino acid sequence set forth in SEQ ID NO: 25, and alight chain variable region that comprises a CDR-L1 that comprises theamino acid sequence set forth in SEQ ID NO: 29), a CDR-L2 that comprisesthe amino acid sequence set forth in SEQ ID NO: 31, and a CDR-L3 thatcomprises the amino acid sequence set forth in SEQ ID NO: 33. In anembodiment of the invention, the further antigen-binding protein (e.g.,antibody or fragment) comprises a heavy chain variable region thatcomprises (i) a CDR-H1 that comprises the amino acid sequence set forthin SEQ ID NO: 5, a CDR-H2 that comprises the amino acid sequence setforth in SEQ ID NO: 7; and a CDR-H3 that comprises the amino acidsequence set forth in SEQ ID NO: 9; and a light chain variable regionthat comprises a CDR-L1 that comprises the amino acid sequence set forthin SEQ ID NO: 13, a CDR-L2 that comprises the amino acid sequence setforth in SEQ ID NO: 15, and a CDR-L3 that comprises the amino acidsequence set forth in SEQ ID NO: 17; (ii) a heavy chain variable regionthat comprises a CDR-H1 that comprises the amino acid sequence set forthin SEQ ID NO: 37, a CDR-H2 that comprises the amino acid sequence setforth in SEQ ID NO: 39, and a CDR-H3 that comprises the amino acidsequence set forth in SEQ ID NO: 41; and a light chain variable regionthat comprises a CDR-L1 that comprises the amino acid sequence set forthin SEQ ID NO: 45, a CDR-L2 that comprises the amino acid sequence setforth in SEQ ID NO: 47, and a CDR-L3 that comprises the amino acidsequence set forth in SEQ ID NO: 49; (iii) a heavy chain variable regionthat comprises a CDR-H1 that comprises the amino acid sequence set forthin SEQ ID NO: 53, a CDR-H2 that comprises the amino acid sequence setforth in SEQ ID NO: 55, a CDR-H3 that comprises the amino acid sequenceset forth in SEQ ID NO: 57; and a light chain variable region thatcomprises a CDR-L1 that comprises the amino acid sequence set forth inSEQ ID NO: 61, a CDR-L2 that comprises the amino acid sequence set forthin SEQ ID NO: 63, and a CDR-L3 that comprises the amino acid sequenceset forth in SEQ ID NO: 65; (iv) a heavy chain variable region thatcomprises a CDR-H1 that comprises the amino acid sequence set forth inSEQ ID NO: 69, a CDR-H2 that comprises the amino acid sequence set forthin SEQ ID NO: 71, and a CDR-H3 that comprises the amino acid sequenceset forth in SEQ ID NO: 73; and a light chain variable region thatcomprises a CDR-L1 that comprises the amino acid sequence set forth inSEQ ID NO: 77, a CDR-L2 that comprises the amino acid sequence set forthin SEQ ID NO: 79, and a CDR-L3 that comprises the amino acid sequenceset forth in SEQ ID NO: 81; (v) a CDR-H1 that comprises the amino acidsequence set forth in SEQ ID NO: 89, a CDR-H2 that comprises the aminoacid sequence set forth in SEQ ID NO: 91, a CDR-H3 that comprises theamino acid sequence set forth in SEQ ID NO: 93, a CDR-L1 that comprisesthe amino acid sequence set forth in SEQ ID NO: 97, a CDR-L2 thatcomprises the amino acid sequence set forth in SEQ ID NO: 99, a CDR-L3L2 that comprises the amino acid sequence set forth in SEQ ID NO: 101;or (vi) a CDR-H1 that comprises the amino acid sequence set forth in SEQID NO: 105, a CDR-H2 that comprises the amino acid sequence set forth inSEQ ID NO: 107, a CDR-H3 that comprises the amino acid sequence setforth in SEQ ID NO: 109; a CDR-L1 that comprises the amino acid sequenceset forth in SEQ ID NO: 97, a CDR-L2 that comprises the amino acidsequence set forth in SEQ ID NO: 99, and a CDR-L3 that comprises theamino acid sequence set forth in SEQ ID NO: 101. In an embodiment of theinvention, the combination includes an optional further therapeuticagent. For example, the further therapeutic agent is, in an embodimentof the invention, one or more anti-C5 antibodies such as H2M11683N;H2M11686N; H4H12159P; H4H12163P; H4H12164P; H4H12166P2; H4H12166P3;H4H12166P4; H4H12166P5; H4H12166P6; H4H12166P7; H4H12166P8; H4H12166P9;H4H12166P10; H4H12167P; H4H12168P; H4H12169P; H4H12176P2; H4H12177P2;H4H12183P2; H2M11682N; H2M11684N; H2M11694N; or H2M11695N or an antibodyor antigen-binding fragment comprising the V_(H) and/or V_(L); and/orCDR-Hs and/or CDR-Ls thereof (See International Patent Application No.PCT/US2017/037226, filed Jun. 13, 2017); or an antigen-binding fragmentof any of the foregoing (which is not a first or second/further antibodyor antigen-binding fragment in the combination). In an embodiment of theinvention, the further therapeutic agent is an eculizumab or coversin(if not already a component of the combination), iron, antithymocyteglobulin, a growth factor, anti-coagulant, a thrombin inhibitor, ananti-inflammatory drug, an antihypertensive, an immunosuppressive agent,a fibrinolytic agent, a lipid-lowering agent, an inhibitor ofhydroxymethylglutaryl CoA reductase, an anti-CD20 agent, an anti-TNFαagent, an anti-seizure agent, a C3 inhibitor, an anti-thrombotic agent,warfarin, aspirin, heparin, phenindione, fondaparinux, idraparinux,argatroban, lepirudin, bivalirudin, or dabigatran, corticosteroids, andnon-steroidal anti-inflammatory drugs, vincristine, cyclosporine A,methotrexate, ancrod, ε-aminocaproic acid, antiplasmin-a1, prostacyclin,defibrotide, rituximab and/or magnesium sulfate.

The present invention provides a bispecific or biparatopicantigen-binding protein (e.g., antibody or antigen-binding fragmentthereof (e.g., an IgG)) comprising a first antigen-binding domain thatbinds to C5 (e.g., human C5) at a first epitope (e.g., oneantigen-binding domain from the H4H12166P antibody) and a secondantigen-binding domain that (i) specifically binds to C5 at a secondepitope which is different from that of the first antigen-binding domainand/or (ii) does not compete with the first antigen-binding domain forbinding to C5 (e.g., one antigen-binding domain from the eculizumab,H4H12161P, H4H12170P, H4H12171P, H4H12175P, H4H12176P2 or H4H12177P2antibody, for example, H4H12176P2×H4H12177P2) or a pharmaceuticallycomposition thereof comprising a pharmaceutically acceptable carrier.The present invention also provides a method for treating or preventinga C5-associated disease or disorder in a subject (e.g., a mammal such asa human) in need of such treatment or prevention or for inhibiting boththe classical and alternative complement pathway (CP and AP,respectively) in a subject comprising administering (e.g.,subcutaneously, intravenously, intradermally, intraperitoneally, orally,intramuscularly or intracranially) an effective amount of a combinationof the present invention (and, optionally, one or more furthertherapeutic agents, e.g., as discussed herein) to the subject. Such adisease or disorder can be, for example, acute respiratory distresssyndrome; adult respiratory distress syndrome; age-related maculardegeneration; allergy; AIport's syndrome; Alzheimer's disease; asthma;asthma; atherosclerosis; atypical hemolytic uremic syndrome; autoimmunediseases; complement activation caused by balloon angioplasty;bronchoconstriction; bullous pemphigoid; burns; C3 glomerulopathy;capillary leak syndrome; chemical injury; chronic obstructive pulmonarydisease; Crohn's disease; diabetes; diabetic macular edema; diabeticnephropathy; diabetic retinopathy; dyspnea; emphysema; epilepsy;fibrogenic dust diseases; frostbite; geographic atrophy; glomerulopathy;Goodpasture's Syndrome; Guillain-Barre Syndrome; complement activationcaused by hemodialysis; hemodialysis complications; hemolytic anemia;hemoptysis; hereditary angioedema; hyperacute allograft rejection;hypersensitivity pneumonitis; immune complex disorders; immunecomplex-associated inflammation; inflammation of autoimmune diseases;inflammatory disorders; inherited CD59 deficiency; injury due to inertdusts and/or minerals; interleukin-2 induced toxicity during IL-2therapy; lupus nephritis; membraneproliferative glomerulonephritis;membranoproliferative nephritis; mesenteric artery reperfusion afteraortic reconstruction; mesenteric artery reperfusion after infectiousdisease; mesenteric artery reperfusion after sepsis; multiple sclerosis;myasthenia gravis; myocardial infarction; neuromyelitis optica;neuromyelitis optica; obesity; ocular angiogenesis; organic dustdiseases; parasitic diseases; Parkinson's disease; paroxysmal nocturnalhemoglobinuria; pneumonia; post-ischemic reperfusion conditions;post-pump syndrome in cardiopulmonary bypass or renal bypass;progressive kidney failure; proteinuric kidney diseases; psoriasis;pulmonary embolisms and infarcts; pulmonary fibrosis; pulmonaryvasculitis; renal ischemia; renal ischemia-reperfusion injury; renaltransplant; rheumatoid arthritis; schizophrenia; smoke injury; stroke;stroke; systemic lupus erythematosus; systemic lupus erythematosusnephritis; thermal injury; thermal injury; traumatic brain injury;uveitis; vasculitis; and xenograft rejection. For example, in anembodiment of the invention, the method comprises administering to thesubject a first antigen-binding protein that specifically binds C5 and asecond antigen-binding protein that specifically binds C5; wherein thefirst and second antigen-binding proteins: (a) bind to distinct,non-overlapping epitopes on C5; and/or (b) do not compete with oneanother for binding to C5, e.g., under conditions which are discussedherein.

Kits of the present invention can also be produced by a methodcomprising the steps of co-packaging the first anti-C5 antigen-bindingprotein (e.g., antibody or fragment); and one or more of said furtheranti-C5 antigen-binding proteins (e.g., polypeptides, antibodies orfragments); and, optionally, one or more further therapeutic agents. Akit which is the product of such a method is also part of the presentinvention.

Co-formulations of the present invention can be produced by a methodcomprising co-formulating (e.g., mixing) said first antigen-bindingprotein (e.g., antibody or fragment); and one or more of said furtherantigen-binding proteins (e.g., polypeptides, antibodies or fragments);and, optionally, one or more further therapeutic agents; and apharmaceutically acceptable carrier into a single pharmaceuticalformulation. A co-formulation which is the product of such a method isalso part of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a graph showing hemolysis of red blood cells in the presenceof serum and an anti-C5 antibody H4H12166P, H4H12170P, H4H12161P,H4H12171P, H4H12175P, H4H12176P2 or H4H12177P2. FIG. 1B is a graphshowing hemolysis of red blood cells in the presence of serum and acombination of H4H12166P+H4H12170P; H4H12166P+H4H12161P;H4H12166P+H4H12171P; H4H12166P+H4H12175P; H4H12166P+H4H12176P2; orH4H12166P+H4H12177P2.

FIG. 2A is a graph showing hemolysis of red blood cells in the presenceof 25% serum and H4H12166P, H4H12161P or H4H12166P+H4H12161P incubatedfor 30 or 120 minutes. FIG. 2B is a graph showing hemolysis of red bloodcells and H4H12166P, H4H12161P or H4H12166P+H4H12161P incubated for 120minutes in the presence of 25% or 48% serum.

FIG. 3. Hemolysis of red blood cells in the presence of serum andH4H12166P; H4H12170P Fab or H4H12166P+H4H12170P Fab incubated for 30 or120 minutes.

FIG. 4. Hemolysis of red blood cells in the presence of serum andH4H12176P2; H4H12177P2; or H4H12176P2+H4H12177P2 incubated for 30minutes.

FIG. 5A is a graph showing hemolysis of red blood cells in the presenceof serum and H4H12170P, H4H12159P; or H4H12170P+H4H12159P, incubated for30 minutes; FIG. 5B is a graph showing hemolysis of red blood cells inthe presence of serum and H4H12175P, H4H12177P2; orH4H12175P+H4H12177P2, incubated for 30 minutes; FIG. 5C is a graphshowing hemolysis of red blood cells in the presence of serum andH4H12176P2, H4H12164P2; or H4H12176P2+H4H12164P2, incubated for 30minutes; FIG. 5D is a graph showing hemolysis of red blood cells in thepresence of serum and H4H12167P, H4H12163P; or H4H12167P+H4H12163P,incubated for 30 minutes.

FIG. 6. Hemolysis of red blood cells in the presence of H4H12166P,H4H12161P alone or in combination with C3 protein.

FIG. 7. Generation of C5a in the presence of H4H12166P; H4H12161P orH4H12166P+H4H12161P.

FIG. 8. Hemolysis assay; alternative complement pathway with 25% normalhuman serum incubated for 30 minutes.

FIG. 9. Hemolysis assay; alternative complement pathway with 25%C5-deficient normal human serum; 145 nM C5 add-back and various ratiosof antibody to C5.

FIG. 10A and FIG. 10B are graphs showing hemolysis assay; alternativecomplement pathway with 25% C5-deficient normal human serum; 125 nM C5add-back and various ratios of bispecific anti-C5 antibody to C5.

FIG. 11. A4F-MALLS analysis of H4H12166P:C5 complexes (mAb:C5::1 μm:1 μmratio) in the absence of a secondary antibody.

FIG. 12. A4F-MALLS analysis of H4H12166P:C5 complexes with secondaryantibodies (mAb2), H4H12175P or H4H12177P2 (mAb1:mAb2:C5::0.5 μm:0.5μm:1 μm ratio).

FIG. 13. A4F-MALLS analysis of H4H12166P: H4H12161P:hC5; H4H12166P:H4H12176P2:hC5; and H4H12176P2: H4H12177P2:hC5 complexes(mAb1:mAb2:C5::0.5 μm:0.5 μm:1 μm ratio).

FIG. 14. A4F-MALLS analysis of H4H12166P:C5 complexes with secondaryantibody, H4H12170P (mAb1:mAb2:C5::0.5 μm:0.5 μm:1 μm ratio).

FIG. 15. A4F-MALLS analysis of H4H12166P:C5 complexes with secondaryantibody, H4H12171P (mAb1:mAb2:C5::0.5 μm:0.5 μm:1 μm ratio).

FIG. 16. A4F-MALLS analysis of H4H12176P2×H4H12177P2:C5 complexes atvarious ratios (mAb:C5::3:1, 1:1 or 1:3).

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are now described. All patents, applications and publicationsmentioned herein are incorporated herein by reference in their entirety.

Definitions

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-timebiomolecular interactions by detection of alterations in proteinconcentrations within a biosensor matrix, for example using the BIACORE™system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction.

Sequence identity refers to the degree to which the amino acids of twopolypeptides are the same at equivalent positions when the two sequencesare optimally aligned. Sequence similarity includes identical residuesand nonidentical, biochemically related amino acids. Examples of groupsof amino acids that have side chains with similar chemical propertiesinclude 1) aliphatic side chains: glycine, alanine, valine, leucine andisoleucine; 2) aliphatichydroxyl side chains: serine and threonine; 3)amide-containing side chains: asparagine and glutamine; 4) aromatic sidechains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains:lysine, arginine, and histidine; 6) acidic side chains: aspartate andglutamate, and 7) sulfur-containing side chains: cysteine andmethionine. Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al. (1992) Science 256: 1443 45, herein incorporated by reference. A“moderately conservative” replacement is any change having a nonnegativevalue in the PAM250 log-likelihood matrix.

A “variant” of a polypeptide, such as an immunoglobulin chain or CDR,refers to a polypeptide comprising an amino acid sequence that is atleast about 70-99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%)identical or similar to a referenced amino acid sequence that is setforth herein; when the comparison is performed by a BLAST algorithmwherein the parameters of the algorithm are selected to give the largestmatch between the respective sequences over the entire length of therespective reference sequences (e.g., expect threshold: 10; word size:3; max matches in a query range: 0; BLOSUM 62 matrix; gap costs:existence 11, extension 1; conditional compositional score matrixadjustment).

A “variant” of a polynucleotide refers to a polynucleotide comprising anucleotide sequence that is at least about 70-99.9% (e.g., 70, 72, 74,75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 99.5, 99.9%) identical to a referenced nucleotidesequence that is set forth herein; when the comparison is performed by aBLAST algorithm wherein the parameters of the algorithm are selected togive the largest match between the respective sequences over the entirelength of the respective reference sequences (e.g., expect threshold:10; word size: 28; max matches in a query range: 0; match/mismatchscores: 1, −2; gap costs: linear).

The following references relate to BLAST algorithms often used forsequence analysis: BLAST ALGORITHMS: Altschul et al. (2005) FEBS J.272(20): 5101-5109; Altschul, S. F., et al., (1990) J. Mol. Biol.215:403-410; Gish, W., et al., (1993) Nature Genet. 3:266-272; Madden,T. L., et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S. F., etal., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997)Genome Res. 7:649-656; Wootton, J. C., et al., (1993) Comput. Chem.17:149-163; Hancock, J. M. et al., (1994) Comput. Appl. Biosci.10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M. O., et al., “A model ofevolutionary change in proteins.” in Atlas of Protein Sequence andStructure, (1978) vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp. 345-352,Natl. Biomed. Res. Found., Washington, D.C.; Schwartz, R. M., et al.,“Matrices for detecting distant relationships.” in Atlas of ProteinSequence and Structure, (1978) vol. 5, suppl. 3.” M. O. Dayhoff (ed.),pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C.; Altschul, S.F., (1991) J. Mol. Biol. 219:555-565; States, D. J., et al., (1991)Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA89:10915-10919; Altschul, S. F., et al., (1993) J. Mol. Evol.36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl.Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob.22:2022-2039; and Altschul, S. F. “Evaluating the statisticalsignificance of multiple distinct local alignments.” in Theoretical andComputational Methods in Genome Research (S. Suhai, ed.), (1997) pp.1-14, Plenum, N.Y.

As used herein, the term “subject” refers to an animal, preferably amammal, more preferably a human, for example, in need of amelioration,prevention and/or treatment of a C5-associated disease or disorder suchas atypical hemolytic uremic syndrome (aHUS) or paroxysmal nocturnalhemoglobinuria (PNH). The term includes human subjects who have or areat risk of having such a disease or disorder.

As used herein a “combination” refers to a collocation of a firstcomponent with is an anti-C5 antigen-binding protein (e.g., antibody orantigen-binding fragment) and one or more further components which is ananti-C5 antigen-binding protein (e.g., antibody, antigen-bindingfragment or polypeptide) (e.g., H4H12166P and one of H4H12161P,H4H12170P, H4H12171P, H4H12175P, H4H12176P2 or H4H12177P2). Such acollocation may be in a single liquid (e.g., aqueous) or dry (e.g.,lyophilized) composition, e.g., a pharmaceutical composition, thatincludes both components. A collocation may be a kit comprising eachcomponent in two or more separate vessels or devices. With regard tocombinations used in connection with methods of treatment or preventionthat are discussed herein, each component, in the combination, can beadministered to a subject at a different time than when the othercomponent is administered; for example, each administration may be givennon-simultaneously (e.g., separately or sequentially) at intervals overa given period of time. Moreover, the separate components may beadministered to a subject by the same or by a different route, e.g.,wherein an anti-C5 antibody is administered subcutaneously and the otheranti-C5 antibody is administered intravenously. In an embodiment of theinvention, the components of a combination are located in a commonmolecule, e.g., a multispecific molecule (e.g. bispecific) that binds toC5 at multiple epitopes. For example, a combination of two anti-C5antibodies or antigen-binding fragments includes a bispecific orbiparatopic antibody or fragment having a first antigen-binding domainthat binds to a first epitope on C5 and a second antigen-binding domainthat binds to a second, different epitope on C5 and/or which does notcompete for binding to C5 with the first antigen-binding domain (e.g.,as discussed further herein).

C5

The present invention relates to combinations including antigen-bindingproteins (e.g., antibodies and antigen-binding fragments) that bind toC5 (“complement component 5” or “complement factor 5”), for example,human C5 (e.g., H4H12166P and one of H4H12161P, H4H12170P, H4H12171P,H4H12175P, H4H12176P2 or H4H12177P2).

The C5 gene encodes a component of the complement system, a part of theinnate immune system that plays an important role in inflammation, hosthomeostasis, and host defense against pathogens. The C5 gene product isproteolytically processed to generate multiple protein products,including the C5 alpha chain, C5 beta chain, C5a anaphylatoxin and C5b.The C5 protein includes the C5 alpha and beta chains which are linked bya disulfide bridge.

The amino acid sequence of full-length C5 protein is exemplified by theamino acid sequence provided in GenBank as accession number NP_001726.2(SEQ ID NO: 1). The term “C5” includes recombinant C5 protein or afragment thereof (e.g. a mature fragment lacking the N-terminal signalpeptide). The term also encompasses C5 protein or a fragment thereofcoupled to, for example, histidine tag, mouse or human Fc, or a signalsequence such as ROR1. The term also includes protein variants thatcomprise a histidine tag at the C-terminal, coupled to amino acidresidues 19-1676 of full-length C5 protein with a R885H change or aR885C change. In an embodiment of the invention, human C5 comprises theamino acid sequence set forth in SEQ ID NO: 1.

Anti-C5 Antibodies, Fragments and Polypeptides

The present invention provides combinations comprising a firstantigen-binding protein (e.g., an antibody or antigen-binding fragmentthereof) that binds specifically to C5 and one or more furtherantigen-binding proteins (e.g., polypeptides or antibodies orantigen-binding fragments thereof) that (i) specifically bind to C5 atan epitope which is different from that of the first antigen-bindingprotein; and/or (ii) do not compete with the first antigen-bindingprotein for binding to C5 (e.g., H4H12166P and one of H4H12161P,H4H12170P, H4H12171P, H4H12175P, H4H12176P2 or H4H12177P2).

An anti-C5 “antigen-binding protein” is a polypeptide or complex of morethan one polypeptide (e.g., a tetrameric IgG antibody) that bindsspecifically to C5 polypeptide, for example, an anti-C5 antibody orantigen-binding fragment.

For example, the present invention includes combinations comprisingantibody H4H12166P (or an antigen-binding fragment thereof) and any oneor more antibodies (or antigen-binding fragments thereof)) selected fromH4H12161P, H4H12170P, H4H12171P, H4H12171P, H4H12175P, H4H12176P2 andH4H12177P2; or Eculizumab (sold as “Soliris”); or the polypeptideOrnithodoros moubata OmCI (or a variant thereof) or EV576 (coversin).See International patent application publication no. WO2004106369 orU.S. patent application publication no. US20170065677 or WO2007028968.

The term “antibody”, as used herein, refers to antigen-binding proteinswhich are immunoglobulin molecules comprising four polypeptide chains,two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds (i.e., “full antibody molecules”), as well as multimersthereof (e.g., IgM) or antigen-binding fragments thereof. Each heavychain includes a heavy chain variable region (“HCVR” or “V_(H)”) and aheavy chain constant region (comprising domains C_(H1), C_(H2) andC_(H3)). Each light chain includes a light chain variable region (“LCVR”or “V_(L)”) and a light chain constant region (C_(L)). The V_(H) andV_(L) regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged, from amino-terminus to carboxy-terminus, in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments ofthe invention, the FRs of the antibody (or antigen binding fragmentthereof) may be identical to the human germline sequences, or may benaturally or artificially modified.

The present invention includes combinations which are multispecific(e.g., bispecific) antigen-binding proteins (e.g., antibodies andantigen-binding fragments thereof) which comprise a firstantigen-binding domain that binds to C5 at a first epitope and a secondantigen-binding domain that (i) specifically binds to C5 at a secondepitope which is different from that of the first antigen-bindingdomain; and/or (ii) do not compete with the first antigen-binding domainfor binding to C5 (or would not compete if the first antigen-bindingdomain and second antigen-binding domain were in separate monospecific(e.g., bivalent IgG) proteins (e.g., antibodies) that were tested forcompetition). A bispecific antigen-binding protein (e.g., antibody) mayalso be called biparatopic insofar as the molecule binds to two epitopeswithin the same antigen (C5). For example, in an embodiment of theinvention, the first antigen-binding domain comprises the heavy andlight chain CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) orthe V_(H) and V_(L) or heavy and light chain of H4H12166P; and thesecond antigen-binding domain comprises the heavy and light chain CDRs(CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) or the V_(H) andV_(L) or heavy and light chain of H4H12161P, H4H12170P, H4H12171P,H4H12175P, H4H12176P2 or H4H12177P2. For example, in an embodiment ofthe invention, the biparatopic antigen-binding protein (e.g., antibodyor antigen-binding fragment) is in a bispecific IgG format (e.g., IgG1,IgG2, IgG3 or IgG4 (e.g., having a Ser228Pro mutation)) that it atetramer comprising two heavy chain/light chain pairs. In an embodimentof the invention, the otherwise biparatopic antigen-binding protein(e.g., antibody or fragment) is appended with one or more additionalantigen-binding immunoglobulins (e.g., an additional C5-bindingimmunoglobulin) or an additional polypeptide (e.g., coversin). Thepresent invention also provides an anti-C5 antigen-binding protein(e.g., antibody or antigen-binding fragment thereof) that is linked witha polypeptide (e.g., coversin) that binds to C5 at a different epitopethan that of the antigen-binding protein (e.g., antibody or fragment)and/or which does not compete with the antigen-binding protein (e.g.,antibody or fragment) for binding to C5. In an embodiment of theinvention, the bispecific antigen-binding protein is a F(ab′)₂ of a fullbispecific antibody (e.g., IgG antibody), e.g., the product of a pepsincleavage of a bispecific IgG antibody. In an embodiment of theinvention, the bispecific antigen-binding protein is abivalent/bispecific scFv that comprises a V_(L) and V_(H) that binds toa first C5 epitope linked, e.g., via linker (e.g., peptide linker), to asecond V_(H) and V_(L) that binds to a second C5 epitope.

Antibodies and antigen-binding fragments discussed herein may beassigned to different classes depending on the amino acid sequences ofthe constant domain of their heavy chains. There are at least five majorclasses of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several ofthese may be further divided into subclasses (isotypes), e.g. IgG1,IgG2, IgG3 and IgG4; IgA1 and IgA2. A human heavy chain constant regioncan be gamma-4 (IgG4) with a Ser228Pro mutation (Schuurman, J et al.,Mol. Immunol. 38: 1-8, 2001). An antibody or antigen-binding fragmentcan comprises a light chain constant region such as a human light chainconstant region (e.g., lambda or kappa human light chain region). Theanti-C5 antibody and antigen-binding fragment V_(H) chains discussedherein may be linked to any of the heavy constant chains discussedherein. The anti-C5 antibody and antigen-binding fragment V_(L) chainsdiscussed herein may be linked to any of the light constant chainsdiscussed herein.

Antibodies and antigen-binding fragments discussed herein may comprise aV_(H) and/or V_(L) set forth herein and a modified Fc. Non-limitingexamples of such Fc modifications include, e.g., a modification atposition 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g.,L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or amodification at position 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or amodification at position 250 and/or 428; or a modification at position307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, themodification comprises a 428L (e.g., M428L) and 434S (e.g., N434S)modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F)modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification;a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Qand 428L modification (e.g., T250Q and M428L); and a 307 and/or 308modification (e.g., 308F or 308P). In yet another embodiment, themodification comprises a 265A (e.g., D265A) and/or a 297A (e.g., N297A)modification. In an embodiment of the invention, a combination comprisesone or more anti-C5 antibodies or antigen-binding fragments comprisingan Fc domain comprising one or more pairs or groups of mutationsselected from the group consisting of: 250Q and 248L (e.g., T250Q andM248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I and Q311I);257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V andN434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and434F (e.g., H433K and N434F). All possible combinations of the foregoingFc domain mutations and other mutations within the antibody variabledomains disclosed herein, are contemplated within the scope of thepresent invention.

The identification of CDRs within an immunoglobulin chain is well knownin the art. The assignment of amino acids to each domain is, in anembodiment of the invention, in accordance with the definitions ofSequences of Proteins of Immunological Interest, Kabat, et al.; NationalInstitutes of Health, Bethesda, Md.; 5^(th) ed.; NIH Publ. No. 91-3242(1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J.Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol.196:901-917 or Chothia, et al., (1989) Nature 342:878-883. Thus, whenreferring to CDRs in a given immunoglobulin chain, said CDRs may, in anembodiment of the invention, be identified using any of the conventionsand methods cited above.

The present invention relates to anti-C5 antibodies and antigen-bindingfragments and polypeptides comprising sequences that are specificallyset forth herein as well as variants thereof. A variant of an anti-C5antibody or fragment disclosed herein may comprise one or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid substitutions, insertionsand/or deletions in the framework and/or CDR regions of the heavy and/orlight chain variable domains (e.g., in any one or more of CDR-H1,CDR-H2, CDR-H3, CDR-L1, CDR-L2 and/or CDR-L3) as compared to thecorresponding specific sequences set forth herein. In an embodiment ofthe invention, a variant of an anti-C5 antibody or fragment has one ormore conservative substitutions; for example, having HCVR, LCVR, and/orCDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 orfewer, 4 or fewer, etc. conservative amino acid substitutions relativeto any of the HCVR, LCVR, and/or CDR amino acid sequences specificallydisclosed herein. In an embodiment of the invention, an anti-C5antibody, fragment or polypeptide is a variant comprising a polypeptide(e.g., an immunoglobulin heavy and/or light chain variable region) aminoacid sequence that is at least about 70-99.9% (e.g., 70, 72, 74, 75, 76,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 99.5, 99.9%) identical or similar to a referenced amino acidsequence that is set forth herein; when the comparison is performed by aBLAST algorithm wherein the parameters of the algorithm are selected togive the largest match between the respective sequences over the entirelength of the respective reference sequences (e.g., expect threshold:10; word size: 3; max matches in a query range: 0; BLOSUM 62 matrix; gapcosts: existence 11, extension 1; conditional compositional score matrixadjustment). In an embodiment of the invention, such a variant retainsthe ability to bind to C5.

In an embodiment of the invention, the anti-C5 antibody orantigen-binding fragment comprises a heavy chain comprising an aminoacid that is at least 70% identical to the amino acid sequence set forthin SEQ ID NO: 3, 19, 35, 51, 67, 82, 84, 87 or 103 and/or a light chaincomprising an amino acid that is at least 70% identical to the aminoacid sequence set forth in SEQ ID NO: 11, 27, 43, 59, 75, 83, 85 or 95.For example, in an embodiment of the invention, while the overallsequence identity of an immunoglobulin chain is less than 100% relativeto that of a reference immunoglobulin chain amino acid sequence, theimmunoglobulin chain comprises CDR1, CDR2 and CDR3 which are 100%identical to that of the CDRs in the reference immunoglobulin chain.

The present invention also relates to combinations including humananti-C5 antigen-binding proteins (e.g., antibodies and antigen-bindingfragments thereof). The term “human antibody”, as used herein, includesantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human mAbs of the invention mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs and in particular CDR3. However, the term “human antibody”, as usedherein, is not intended to include mAbs in which CDR sequences derivedfrom the germline of another mammalian species (e.g., mouse), have beengrafted onto human FR sequences. The term includes antibodiesrecombinantly produced in a nonhuman mammal, or in cells of a non-humanmammal. The term is not intended to include antibodies isolated from orgenerated in a human subject. Methods for generating human antibodies intransgenic mice are known in the art. Any such known methods can be usedin the context of the present invention to make human antibodies thatspecifically bind to C5 protein. Using VELOCIMMUNE® technology (see, forexample, U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals,VELOCIMMUNE®) or any other known method for generating monoclonalantibodies, high affinity chimeric antibodies to C5 can be initiallyisolated having a human variable region and a mouse constant region. TheVELOCIMMUNE® technology involves generation of a transgenic mouse havinga genome comprising human heavy and light chain variable regionsoperably linked to endogenous mouse constant region loci such that themouse produces an antibody comprising a human variable region and amouse constant region in response to antigenic stimulation. The DNAencoding the variable regions of the heavy and light chains of theantibody can be isolated and operably linked to DNA encoding the humanheavy and light chain constant regions. The DNA can then be expressed ina cell capable of expressing the fully human antibody. Generally, aVELOCIMMUNE® mouse is challenged with the antigen of interest, andlymphatic cells (such as B-cells) are recovered from the mice thatexpress antibodies. The lymphatic cells may be fused with a myeloma cellline to prepare immortal hybridoma cell lines, and such hybridoma celllines are screened and selected to identify hybridoma cell lines thatproduce antibodies specific to the antigen of interest. DNA encoding thevariable regions of the heavy chain and light chain may be isolated andlinked to desirable isotypic constant regions of the heavy chain andlight chain. Such an antibody protein may be produced in a cell, such asa CHO cell. Alternatively, DNA encoding the antigen-specific chimericantibodies or the variable domains of the light and heavy chains may beisolated directly from antigen-specific lymphocytes.

The present invention also relates to combinations including recombinantanti-C5 antigen-binding proteins (e.g., antibodies and antigen-bindingfragments thereof). The term “recombinant”, as used herein, refers toantibodies or antigen-binding fragments thereof of the inventioncreated, expressed, isolated or obtained by technologies or methodsknown in the art as recombinant DNA technology which include, e.g., DNAsplicing and transgenic expression. The term refers to antigen-bindingproteins, such as antibodies, expressed in a non-human mammal (includingtransgenic non-human mammals, e.g., transgenic mice), or a cell (e.g.,CHO cells) expression system or isolated from a recombinantcombinatorial human antibody library. See e.g., U.S. Pat. Nos.4,816,567; 6,331,415 and 7,923,221.

The present invention also relates to combinations including blocking orneutralizing anti-C5 antigen-binding proteins (e.g., antibodies andantigen-binding fragments thereof and polypeptides). A “blocking” or“neutralizing” antigen-binding protein, e.g., antibody, fragment orpolypeptide, as used herein (or an antibody, fragment or polypeptidethat “neutralizes C5 activity” or an “antagonist” antibody, fragment orpolypeptide), is intended to refer to a protein whose binding to C5results in inhibition of at least one biological activity of C5. Forexample, an antibody of the invention may prevent or blockcomplement-mediated hemolysis (e.g., of a red blood cell), e.g., by theclassical pathway or the alternative pathway.

The present invention also relates to combinations including anti-C5antigen-binding proteins which are antigen-binding fragments of anantibody. The terms “antigen-binding portion” of an antibody,“antigen-binding fragment” of an antibody, and the like, as used herein,include a naturally occurring, enzymatically obtainable, synthetic, orgenetically engineered polypeptide or glycoprotein, other than a fullantibody, that specifically binds an antigen to form a complex. Theterms “antigen-binding fragment” of an antibody, or “antibody fragment”,as used herein, refers to one or more fragments of an antibody thatretain the ability to bind to C5 protein. Antigen-binding fragmentsinclude (i) Fab fragments; (ii) F(ab′)₂ fragments; (iii) Fd fragments;(iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAbfragments; and (vii) minimal recognition units including the amino acidresidues that mimic the hypervariable region of an antibody (e.g., anisolated complementarity determining region (CDR) such as a CDR3peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineeredmolecules, such as domain-specific antibodies, single domain antibodies,domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies,diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g.,monovalent nanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein. In certain embodiments, the term “antigen-binding fragment”refers to a polypeptide fragment of a multi-specific antigen-bindingmolecule. Antigen-binding fragments of an antibody may be derived, e.g.,from full antibody molecules using any suitable standard techniques suchas proteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and (optionally) constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

In an embodiment of the invention, an antigen-binding fragment of anantibody comprises at least one variable domain. The variable domain maybe of any size or amino acid composition and will generally comprise atleast one CDR, which is adjacent to or in frame with one or moreframework sequences. In antigen-binding fragments having a V_(H) domainassociated with a V_(L) domain, the V_(H) and V_(L) domains may besituated relative to one another in any suitable arrangement. Forexample, the variable region may be dimeric and contain V_(H)-V_(H),V_(H)-V_(L) or V_(L)-V_(L) dimers. Alternatively, the antigen-bindingfragment of an antibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibodyincludes at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include:

(i) V_(H)-C_(H1);(ii) V_(H)-C_(H2);(iii) V_(H)-C_(H3);(iv) V_(H)-C_(H1)-C_(H2);(v) V_(H)-C_(H1)-C_(H2)-C_(H3);(vi) V_(H)-C_(H2)-C_(H3);(vii) V_(H)-C_(L);(viii) V_(L)-C_(H1);(ix) V_(L)-C_(H2);(x) V_(L)-C_(H3);(xi) V_(L)-C_(H1)-C_(H2),(xii) V_(L)-C_(H1)-C_(H2)-C_(H3);(xiii) V_(L)-C_(H2)-C_(H3); and(xiv) V_(L)-C_(L).In any configuration of variable and constant domains, including any ofthe exemplary configurations listed above, the variable and constantdomains may be either directly linked to one another or may be linked bya full or partial hinge or linker region. A hinge region may consist ofat least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, whichresult in a flexible or semi-flexible linkage between adjacent variableand/or constant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

The present invention also relates to combinations includingmultispecific (e.g., bispecific) antigen-binding proteins (e.g.,antibodies, antigen-binding fragments or polypeptides). The termmultispecific includes the term multiparatopic (and biparatopic).Multiparatopic molecules bind to multiple epitopes within the sameantigen. A multispecific antigen-binding fragment of an antibody willtypically comprise at least two different variable domains, wherein eachvariable domain is capable of specifically binding to a differentantigen or to a different epitope on the same antigen (e.g.,biparatopic). A biparatopic IgG antibody comprises two differentheavy/light chain pairs that bind to two different epitopes within C5.

“Isolated” antigen-binding proteins, e.g., antibodies or antigen-bindingfragments thereof, polypeptides, polynucleotides and vectors, are atleast partially free of other biological molecules from the cells orcell culture from which they are produced. Such biological moleculesinclude nucleic acids, proteins, other antibodies or antigen-bindingfragments, lipids, carbohydrates, or other material such as cellulardebris and growth medium. Isolated antibodies or antigen-bindingfragments thereof, polypeptides, polynucleotides and vectors may furtherbe at least partially free of expression system components such asbiological molecules from a host cell or of the growth medium thereof.Generally, the term “isolated” is not intended to refer to a completeabsence of such biological molecules or to an absence of water, buffers,or salts or to components of a pharmaceutical formulation that includesthe antibodies or antigen-binding fragments thereof, polypeptides,polynucleotides and/or vectors.

The term “specifically binds,” or “binds specifically to”, or the like,means that an antigen-binding protein, e.g., antibody or antigen-bindingfragment thereof, forms a complex with an antigen that is relativelystable under physiologic conditions. Specific binding can becharacterized by an equilibrium dissociation constant of at least about1×10⁻⁸ M or a lower number (e.g., a smaller K_(D) denotes a tighterbinding), for example, at least 10⁻⁹M or 10⁻¹⁰ M, or at least 1.29×10⁻¹⁰M for binding to C5 at 25° C. as measured by SPR or at least 2.62×10⁻¹°M for binding to C5 at 37° C. as measured by SPR.

The term “anti-C5” refers to an antigen-binding protein, e.g., anantibody, antigen-binding fragment, polypeptide or other molecule, thatspecifically binds to C5 polypeptide or an immunogenic fragment thereof.

Methods for determining whether two molecules specifically bind are wellknown in the art and include, for example, equilibrium dialysis, surfaceplasmon resonance, and the like. As described herein, antibodies havebeen identified by surface plasmon resonance, e.g., BIACORE™, which bindspecifically to C5.

In an embodiment of the invention, antigen-binding proteins, e.g., anantibody or antibody fragment of the invention, may be conjugated to amoiety such a ligand or a therapeutic moiety (“immunoconjugate”), asecond anti-C5 antibody, or any other therapeutic moiety useful fortreating a C5-associated disease or disorder. In an embodiment of theinvention, an anti-C5 antigen-binding protein, e.g., antibody orantigen-binding fragment thereof as set forth herein, is conjugated tocoversin polypeptide.

A selection of anti-C5 antibodies and antigen-binding fragments, whereineach of which do not compete with H4H12166P for C5 binding, includesH4H12161P, H4H12170P, H4H12171P, H4H12175P, H4H12176P2 and H4H12177P2.The present invention includes combinations including any two or more ofthese antibodies or antigen-binding fragments thereof. As discussedabove, a combination may be a multispecific (e.g., bispecific) antibodycomprising a heavy and light chain of one such antibody and the heavyand light chain of another such antibody. For example, the scope of thepresent invention includes a bispecific antibody or antigen-bindingfragment thereof comprising a combination of heavy chain immunoglobulinand light chain immunoglobulin taken from any of H4H12161P, H4H12166P,H4H12170P, H4H12171P, H4H12175P, H4H12176P2 and H4H12177P2 to form anantigen-binding domain and a combination of a different heavy chainimmunoglobulin and a different light chain immunoglobulin taken from anyof H4H12161P, H4H12166P, H4H12170P, H4H12171P, H4H12175P, H4H12176P2 andH4H12177P2 to form a different antigen-binding domain. A summary oflight and heavy chain combinations making up some bispecific antibodiesand antigen-binding fragments of the present invention is set forthbelow in Table A. An “X” indicates a bispecific antibody including anantigen-binding domain from the antibody on the horizontal axis and anantigen-binding domain from an antibody on the vertical axis (e.g., aH4H12176P2×H4H12177P2 bispecific antibody). As used herein bispecificantibodies may be referred to as “A×B” wherein A is the antigen-bindingdomain of a first antibody and B is the antigen-binding domain from asecond, different antibody.

TABLE A Exemplary bispecific antibody chain combinations* H4H12161PH4H12166P H4H12170P H4H12171P H4H12175P H4H12176P2 H4H12177P2 H4H12161PH4H12166P x H4H12170P x x H4H12171P x x x H4H12175P x x x x H4H12176P2 xx x x x H4H12177P2 x x x x x *Combinations comprising the two individualantigen-binding proteins indicated in Table A with an “x” are also partof the present invention.

“H4H12161P”, “H4H12166P”, “H4H12170P”, “H4H12171P”, “H4H12175P”,“H4H12176P2” and “H4H12177P2”, for example, refer to antibodies andantigen-binding fragments thereof (or, in the context of a bispecificantibody or antigen-binding fragment, to an antigen-binding domainthereof) that comprise the heavy chain or V_(H) (or a variant thereof)and light chain or V_(L) (or a variant thereof) as set forth below; orthat comprise a V_(H) that comprises the CDRs thereof (CDR-H1 (or avariant thereof), CDR-H2 (or a variant thereof) and CDR-H3 (or a variantthereof)) and a V_(L) that comprises the CDRs thereof (CDR-L1 (or avariant thereof), CDR-L2 (or a variant thereof) and CDR-L3 (or a variantthereof)), e.g., wherein the immunoglobulin chains, variable regionsand/or CDRs comprise the specific amino acid sequences described below.Such nomenclature may be used herein to refer to other antibodies andantigen binding fragments and antigen-binding domains thereof disclosedin WO2017/218515.

The present invention, thus, includes, but is not limited to,multispecific (e.g., bispecific or biparatopic) antibodies andantigen-binding fragments thereof including “H4H12161P×H4H12177P2”;“H4H12166P×H4H12177P2”; “H4H12170P×H4H12177P2”; “H4H12171P×H4H12177P2”;“H4H12176P2×H4H12177P2”; “H4H12176P2×H4H12161P”; “H4H12176P2×H4H12166P”;“H4H12176P2×H4H12170P”; “H4H12176P2×H4H12171P”; “H4H12176P2×H4H12175P”;“H4H12175P×H4H12161P”; “H4H12175P×H4H12166P”; “H4H12175P×H4H12170P”;“H4H12175P×H4H12171P”; “H4H12171P×H4H12161P”; “H4H12171P×H4H12166P”;“H4H12171P×H4H12170P”; “H4H12170P×H4H12161P”; “H4H12170P×H4H12166P”; and“H4H12166P×H4H12161P”.

For example, the multispecific (e.g., bispecific or biparatopic)antibody or antigen-binding fragment, H4H12176P2×H4H12177P2, comprises:

a first antigen binding domain comprising:(1)a heavy chain immunoglobulin or variable region thereof that comprisesCDR-H1, CDR-H2 and CDR-H3 of a heavy chain immunoglobulin or variableregion thereof that comprises the amino acid sequence set forth in SEQID NO: 87; or a variant thereof; anda light chain immunoglobulin or variable region thereof that comprisesCDR-L1, CDR-L2 and CDR-L3 of a light chain immunoglobulin or variableregion thereof that comprises the amino acid sequence set forth in SEQID NO: 95 or a variant thereof; anda second antigen binding domain comprisinga heavy chain immunoglobulin or variable region thereof that comprisesCDR-H1, CDR-H2 and CDR-H3 of a heavy chain immunoglobulin or variableregion thereof that comprises the amino acid sequence set forth in SEQID NO: 103; or a variant thereof; anda light chain immunoglobulin or variable region thereof that comprisesCDR-L1, CDR-L2 and CDR-L3 of a light chain immunoglobulin or variableregion thereof that comprises the amino acid sequence set forth in SEQID NO: 95 or a variant thereof;or(2)a first antigen binding domain comprising:a heavy chain variable region comprisinga CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 89;a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 91;anda CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 93;and a light chain variable region comprisinga CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 97;a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 99;anda CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 101;and a second antigen binding domain comprising:a heavy chain variable region comprisinga CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 105;a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 107;anda CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 109;and a light chain variable region comprisinga CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 97;a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 99;anda CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 101;or(3)a first antigen binding domain comprising:(a) a heavy chain immunoglobulin or variable region thereof comprisingthe CDR-H1, CDR-H2 and CDR-H3 of a heavy chain immunoglobulin orvariable region thereof comprising an amino acid sequence set forth inSEQ ID NO: 87 and at least 90% amino acid sequence identity to the aminoacid sequence set forth in SEQ ID NO: 87; and/or(b) an light chain immunoglobulin or variable region thereof comprisingthe CDR-L1, CDR-L2 and CDR-L3 of a light chain immunoglobulin orvariable region thereof comprising an amino acid sequence set forth inSEQ ID NO: 95 and at least 90% amino acid sequence identity to the aminoacid sequence set forth in SEQ ID NO: 95.a second antigen binding domain comprising:(a) a heavy chain immunoglobulin or variable region thereof comprisingthe CDR-H1, CDR-H2 and CDR-H3 of a heavy chain immunoglobulin orvariable region thereof comprising an amino acid sequence set forth inSEQ ID NO: 103 and at least 90% amino acid sequence identity to theamino acid sequence set forth in SEQ ID NO: 103; and/or(b) an light chain immunoglobulin or variable region thereof comprisingthe CDR-L1, CDR-L2 and CDR-L3 of a light chain immunoglobulin orvariable region thereof comprising an amino acid sequence set forth inSEQ ID NO: 95 and at least 90% amino acid sequence identity to the aminoacid sequence set forth in SEQ ID NO: 95;or(4)a first antigen binding domain comprising:a heavy chain immunoglobulin or variable region thereof comprising anamino acid sequence having at least 90% (e.g., 100%) amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 87; and/ora light chain immunoglobulin or variable region thereof comprising anamino acid sequence having at least 90% (e.g., 100%)amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 95; anda second antigen binding domain comprising:a heavy chain immunoglobulin or variable region thereof comprising anamino acid sequence having at least 90% (e.g., 100%)amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 103; and/ora light chain immunoglobulin or variable region thereof comprising anamino acid sequence having at least 90% (e.g., 100%)amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 95.*Analogous multispecific antigen-binding protein embodiments comprisingother combinations of immunoglobulin chains which are set forth hereinare also part of the present invention.

Multispecific (e.g., bispecific) antigen-binding proteins of the presentinvention include two or more different antigen-binding domain which areselected from any of the anti-C5 antibodies set forth in WO2017/218515,e.g., H2M11683N; H2M11686N; H4H12159P; H4H12161P; H4H12163P; H4H12164P;H4H12166P; H4H12166P2; H4H12166P3; H4H12166P4; H4H12166P5; H4H12166P6;H4H12166P7; H4H12166P8; H4H12166P9; H4H12166P10; H4H12167P; H4H12168P;H4H12169P; H4H12170P; H4H12171P; H4H12175P; H4H12176P2; H4H12177P2;H4H12183P2; H2M11682N; H2M11684N; H2M11694N or H2M11695N—in anembodiment of the invention, the antigen binding domains are taken fromnon-competing antibodies in this list; in an embodiment of theinvention, the antigen binding domains are taken from competingantibodies in this list. See Table 1 herein. WO2017/218515 is hereinincorporated by reference in its entirety. In an embodiment of theinvention, an antigen-binding domain is taken from eculizumab orALXN1210 (Ravulizumab).

H4H12161P V_(H) domain (DNA):GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTTGGTCCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACCACTATATGGACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGACTGGATTGGCCGTATTAGAAACAAAGCTAACGCTTATAACACAGAATACGCCGCGTCTGTGAGAGGCAGATTCACCATCTCAAGAGATGATTCACAGAATTTACTGTATCTGCAAATGAACAGCCTGAAAACCGATGACACGGCCGTATATTATTGTGTTAGAGTCTGGAACTACGCCTACTTCGCTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO: 2) V_(H) domain (Polypeptide):EVQLVESGGDLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLDWIGRIRNKANAYNTEYAASVRGRFTISRDDSQNLLYLQMNSLKTDDTAVYYCVRVWNYAYFAMDVWGQGTTVTVSS (SEQ ID NO: 3)CDR-H1 (DNA): GGA TTC ACC TTC AGT GAC CAC TAT (SEQ ID NO: 4)CDR-H1 (Polypeptide): G F T F S D H Y(SEQ ID NO: 5 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) CDR-H2 (DNA): ATT AGA AAC AAA GCT AAC GCT TAT AAC ACA(SEQ ID NO: 6) CDR-H2 (Polypeptide): I R N K A N A Y N T(SEQ ID NO: 7 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) CDR-H3 (DNA):GTT AGA GTC TGG AAC TAC GCC TAC TTC GCT ATG GAC GTC (SEQ ID NO: 8)CDR-H3 (Polypeptide): V R V W N Y A Y F A M D V(SEQ ID NO: 9 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) V_(L) domain (DNA):GACATCCAGATGACCCAGTCTCCATCCTCCCTATCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGTCAAGTCAGAACATTGGAATCTTTTTAAACTGGTATCAACAAAAACCAGGGGAAGCCCCTAACCTCCTGATCTCCGCTGCATCCAGTTTACACAGTGGGGTCCCTTCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTTCACTCTCACCATCGGCAGTCTGCAGCCTGAAGATTTTGCGACTTACTACTGTCAACAGACGTACAATACCATATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA (SEQ ID NO: 10) V_(L) domain (Polypeptide):DIQMTQSPSSLSASVGDRVTITCRSSQNIGIFLNWYQQKPGEAPNLLISAASSLHSGVPSRFSGSGSGTDFTLTIGSLQPEDFATYYCQQTYNTIFTFGPGTKVDIK (SEQ ID NO: 11) CDR-L1 (DNA):CAG AAC ATT GGA ATC TTT (SEQ ID NO: 12) CDR-L1 (Polypeptide):Q N I G I F(SEQ ID NO: 13 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) CDR-L2 (DNA): GCT GCA TCC (SEQ ID NO: 14)CDR-L2 (Polypeptide): A A S(SEQ ID NO: 15 (or a variant thereof having a point mutation or point deletion))CDR-L3 (DNA): CAA CAG ACG TAC AAT ACC ATA TTC ACT (SEQ ID NO: 16)CDR-L3 (Polypeptide): Q Q T Y N T I F T(SEQ ID NO: 17 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) H4H12166P V_(H) domain (DNA):CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGACTCCGTCAGTAGTTCCTACTGGACCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGCTATATCTATTACAGTGGGAGTTCCAACTACAACCCCTCCCTCAAGAGTCGAGCCACCATTTCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCTGCGGACACGGCCGTATATTACTGTGCGAGAGAAGGGAACGTGGATACAACTATGATATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 18)V_(H) domain (Polypeptide):QVQLQESGPGLVKPSETLSLTCTVSGDSVSSSYWTWIRQPPGKGLEWIGYIYYSGSSNYNPSLKSRATISVDTSKNQFSLKLSSVTAADTAVYYCAREGNVDTTMIFDYWGQGTLVTVSS (SEQ ID NO: 19)Heavy immunoglobulin chain hIgG4 (M428L N434S)QVQLQESGPGLVKPSETLSLTCTVSGDSVSSSYWTWIRQPPGKGLEWIGYIYYSGSSNYNPSLKSRATISVDTSKNQFSLKLSSVTAADTAVYYCAREGNVDTTMIFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLGK(SEQ ID NO: 82) CDR-H1 (DNA): GGT GAC TCC GTC AGT AGT TCC TAC(SEQ ID NO: 20) CDR-H1 (Polypeptide): G D S V S S S Y(SEQ ID NO: 21 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) CDR-H2 (DNA): ATC TAT TAC AGT GGG AGT TCC (SEQ ID NO: 22)CDR-H2 (Polypeptide): I Y Y S G S S(SEQ ID NO: 23 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) CDR-H3 (DNA):GCG AGA GAA GGG AAC GTG GAT ACA ACT ATG ATA TTT GAC TAC (SEQ ID NO: 24)CDR-H3 (Polypeptide): A R E G N V D T T M I F D Y(SEQ ID NO: 25 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) V_(L) domain (DNA):GCCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAACAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGCTGCATCCAGTTTACAAAGTGGGGTCCCATCGAGGTTCGCCGGCCGTGGATCTGGCACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAAGATTTCAATTACCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (SEQ ID NO: 26) V_(L) domain (Polypeptide):AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFAGRGSGTDFTLTISSLQPEDFATYYCLQDFNYPWTFGQGTKVEIK (SEQ ID NO: 27)Light immunoglobulin chain (kappa)AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFAGRGSGTDFTLTISSLQPEDFATYYCLQDFNYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 83) CDR-L1 (DNA): CAG GGC ATT AGA AAT GAT (SEQ ID NO: 28)CDR-L1 (Polypeptide): Q G I R N D(SEQ ID NO: 29 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) CDR-L2 (DNA): GCT GCA TCC (SEQ ID NO: 30)CDR-L2 (Polypeptide): A A S(SEQ ID NO: 31 (or a variant thereof having a point mutation or point deletion))CDR-L3 (DNA): CTA CAA GAT TTC AAT TAC CCG TGG ACG (SEQ ID NO: 32)CDR-L3 (Polypeptide): L Q D F N Y P W T(SEQ ID NO: 33 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) H4H12170P V_(H) domain (DNA):CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTGGTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTTATATGGCTTGATGGAAGTAATGACTACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGTTATATCTGCAAATGAACAGACTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGCCCGGTTGCTGCTATACCCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 34)V_(H) domain (Polypeptide):QVQLVESGGGVVQPGRSLRLSCAASGFTFSGYGMHWVRQAPGKGLEWVALIWLDGSNDYYADSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCARDGPVAAIPDYWGQGTLVTVSS (SEQ ID NO: 35)Heavy immunoglobulin chain (IgG4):QVQLVESGGGVVQPGRSLRLSCAASGFTFSGYGMHWVRQAPGKGLEWVALIWLDGSNDYYADSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCARDGPVAAIPDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP(SEQ ID NO: 84) CDR-H1 (DNA): GGA TTC ACC TTC AGT GGT TAT GGC(SEQ ID NO: 36) CDR-H1 (Polypeptide): G F T F S G Y G(SEQ ID NO: 37 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) CDR-H2 (DNA): ATA TGG CTT GAT GGA AGT AAT GAC(SEQ ID NO: 38) CDR-H2 (Polypeptide): I W L D G S N D(SEQ ID NO: 39 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) CDR-H3 (DNA):GCG AGA GAT GGC CCG GTT GCT GCT ATA CCC GAC TAC (SEQ ID NO: 40)CDR-H3 (Polypeptide): A R D G P V A A I P D Y(SEQ ID NO: 41 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) V_(L) domain (DNA):GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGGTGGTTGGCCTGGTATCAGCTGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGACTTCACTCTCACCATCAGCAGCCTGCAACCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATACTTATTCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 42) V_(L) domain (Polypeptide):DIQMTQSPSTLSASVGDRVTITCRASQSISRWLAWYQLKPGKAPKLLIYKASSLESGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQYNTYSYTFGQGTKLEIK (SEQ ID NO: 43)Light immunoglobulin chain (kappa)DIQMTQSPSTLSASVGDRVTITCRASQSISRWLAWYQLKPGKAPKLLIYKASSLESGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQYNTYSYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 85) CDR-L1 (DNA): CAG AGT ATT AGT AGG TGG (SEQ ID NO: 44)CDR-L1 (Polypeptide): Q S I S R W(SEQ ID NO: 45 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) CDR-L2 (DNA): AAG GCG TCT (SEQ ID NO: 46)CDR-L2 (Polypeptide): K A S(SEQ ID NO: 47 (or a variant thereof having a point mutation or point deletion))CDR-L3 (DNA): CAA CAG TAT AAT ACT TAT TCG TAC ACT (SEQ ID NO: 48)CDR-L3 (Polypeptide): Q Q Y N T Y S Y T(SEQ ID NO: 49 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) H4H12171P V_(H) domain (DNA):GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGAATATGGCATGACTTGGGTCCGCCAAGTTCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTATTACTTGGAATGGTGGTTTCACAGATTATACAGACTCTGTGAAGGGCCGATTCACCAGCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGAGATGGATATAGCAGCTCGTGGGGGGCTTATGATATATGGGGCCAAGGGACAATGGTCACCGTCTCTTCA (SEQ ID NO: 50)V_(H) domain (Polypeptide):EVQLVESGGGVVRPGGSLRLSCAASGFTFDEYGMTWVRQVPGKGLEWVSGITWNGGFTDYTDSVKGRFTSSRDNAKNSLYLQMNSLRAEDTALYYCARDGYSSSWGAYDIWGQGTMVTVSS (SEQ ID NO: 51)CDR-H1 (DNA): GGA TTC ACC TTT GAT GAA TAT GGC (SEQ ID NO: 52)CDR-H1 (Polypeptide): G F T F D E Y G(SEQ ID NO: 53 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) CDR-H2 (DNA): ATT ACT TGG AAT GGT GGT TTC ACA(SEQ ID NO: 54) CDR-H2 (Polypeptide): I T W N G G F T(SEQ ID NO: 55 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) CDR-H3 (DNA):GCG AGA GAT GGA TAT AGC AGC TCG TGG GGG GCT TAT GAT ATA (SEQ ID NO: 56)CDR-H3 (Polypeptide): A R D G Y S S S W G A Y D I(SEQ ID NO: 57 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) V_(L) domain (DNA):GACATCCAGATGACCCAGTCTCCATCATCCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCACCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATTAAGGTTCAGTGGCAGTGGATCTGGGACTGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAAGTTATTTCTGTCAACAGAGTTACAGTACCCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 58) V_(L) domain (Polypeptide):DIQMTQSPSSLSASVGDRVTITCRASQSISTYLNWYQQKPGKAPKLLIYAASSLQSGVPLRFSGSGSGTDFTLTISSLQPEDFASYFCQQSYSTPYTFGQGTKLEIK (SEQ ID NO: 59) CDR-L1 (DNA):CAG AGC ATT AGC ACC TAT (SEQ ID NO: 60) CDR-L1 (Polypeptide):Q S I S T Y(SEQ ID NO: 61 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) CDR-L2 (DNA): GCT GCA TCC (SEQ ID NO: 62)CDR-L2 (Polypeptide): A A S(SEQ ID NO: 63 (or a variant thereof having a point mutation or point deletion))CDR-L3 (DNA): CAA CAG AGT TAC AGT ACC CCG TAC ACT (SEQ ID NO: 64)CDR-L3 (Polypeptide): Q Q S Y S T P Y T(SEQ ID NO: 65 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) H4H12175P V_(H) domain (DNA):GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGTCAAGCTCCAGGGAAGGGTCTGGAGTGGGTCTCTCTTATTAGTGGAGATGGTGGTAACACATACTATGCAGACTCTGTGAAGGGCCGACTCACCATCTCCAGAGACAACAGCAAAAACTCCCTGTATCTGCAAATGAACAGTCTGAGAACAGAGGACACCGCCTTATATTACTGTGCAAAAGATAAGGGCTGGAACTTCGGTTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA (SEQ ID NO: 66)V_(H) domain (Polypeptide):EVQLVESGGGVVQPGGSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSLISGDGGNTYYADSVKGRLTISRDNSKNSLYLQMNSLRTEDTALYYCAKDKGWNFGYFDLWGRGTLVTVSS (SEQ ID NO: 67)CDR-H1 (DNA): GGA TTC ACC TTT AAT GAT TAT GCC (SEQ ID NO: 68)CDR-H1 (Polypeptide): G F T F N D Y A(SEQ ID NO: 69 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) CDR-H2 (DNA): ATT AGT GGA GAT GGT GGT AAC ACA(SEQ ID NO: 70) CDR-H2 (Polypeptide): I S G D G G N T(SEQ ID NO: 71 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) CDR-H3 (DNA)GCA AAA GAT AAG GGC TGG AAC TTC GGT TAC TTC GAT CTC (SEQ ID NO: 72)CDR-H3 (Polypeptide): A K D K G W N F G Y F D L(SEQ ID NO: 73 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) V_(L) domain (DNA):GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTACATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAACATTGACACCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGATGCATCCAGTTTACAAAGTGGGGTCCCATCACGGTTCAGTGGCAGCGGATCTGGGACAGATTTCACTCTCACCATCACCAGTCTGCAACCTGAAGATTTTGCCACTTACTACTGTCAACAGAATGACAATATTCTTCACCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO: 74) V_(L) domain (Polypeptide):DIQMTQSPSSLSTSVGDRVTITCRASQNIDTYLNWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTITSLQPEDFATYYCQQNDNILHPLTFGGGTKVEIK (SEQ ID NO: 75) CDR-L1 (DNA):CAG AAC ATT GAC ACC TAT (SEQ ID NO: 76) CDR-L1 (Polypeptide):Q N I D T Y(SEQ ID NO: 77 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) CDR-L2 (DNA): GAT GCA TCC (SEQ ID NO: 78)CDR-L2 (Polypeptide): D A S(SEQ ID NO: 79 (or a variant thereof having a point mutation or point deletion))CDR-L3 (DNA): CAA CAG AAT GAC AAT ATT CTT CAC CCT CTC ACT(SEQ ID NO: 80) CDR-L3 (Polypeptide): Q Q N D N I L H P L T(SEQ ID NO: 81 (or a variant thereof having 1, 2, 3 or 4 point mutations and/or pointdeletions)) H4H12176P2 V_(H) domain (DNA):GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAACCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCCACTCTAATAGATATTGGATGGACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAGCAAGATGGAAGTGAGGAAAACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTTTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCGAAGCACCTCGTGGGTCCCTTACTGGTTCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA(SEQ ID NO: 86) V_(H) domain (Polypeptide):EVQLVESGGGLVQPGGSLRLSCAASGFHSNRYWMDWVRQAPGKGLEWVANIKQDGSEENYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRSTSWVPYWFFDLWGRGTLVTVSS (SEQ ID NO: 87)CDR-H1 (DNA): GGA TTC CAC TCT AAT AGA TAT TGG (SEQ ID NO: 88)CDR-H1 (Polypeptide): G F H S N R Y W (SEQ ID NO: 89) CDR-H2 (DNA):ATA AAG CAA GAT GGA AGT GAG GAA (SEQ ID NO: 90) CDR-H2 (Polypeptide):I K Q D G S E E (SEQ ID NO: 91) CDR-H3 (DNA):GCG AGA GAT CGA AGC ACC TCG TGG GTC CCT TAC TGG TTC TTC GAT CTC(SEQ ID NO: 92) CDR-H3 (Polypeptide): A R D R S T S W V P Y W F F D L(SEQ ID NO: 93) V_(L) domain (DNA):GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA (SEQ ID NO: 94) V_(L) domain (Polypeptide):DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (SEQ ID NO: 95) CDR-L1 (DNA):CAG AGC ATT AGC AGC TAT (SEQ ID NO: 96) CDR-L1 (Polypeptide):Q S I S S Y (SEQ ID NO: 97) CDR-L2 (DNA): GCT GCA TCC (SEQ ID NO: 98)CDR-L2 (Polypeptide): A A S (SEQ ID NO: 99) CDR-L3 (DNA):CAA CAG AGT TAC AGT ACC CCT CCG ATC ACC (SEQ ID NO: 100)CDR-L3 (Polypeptide): Q Q S Y S T P P I T (SEQ ID NO: 101) H4H12177P2V_(H) domain (DNA):GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTACAGCGGGGGGAGTCCCTGAGACTCTCCTGTTCAGCCTCTGACTTCATCTTTAAAGATTATGCCATGTACTGGGTCCGTCAAATTCCAGGGAAGGGTCTAGAGTGGATCTCTCTTATTAGTGGTGATGGTGACACTACATGGTATGGAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACAACGAAAACTCCCTCTTTCTGCAAATGAACGATCTGAGAACTGAGGACACCGCCATGTACTACTGTGCAAGAGATATGGGGTGGAACTTCTTTCAGTTGCAATACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 102)V_(H) domain (Polypeptide):EVQLVESGGGVVQRGESLRLSCSASDFIFKDYAMYWVRQIPGKGLEWISLISGDGDTTWYGDSVKGRFTISRDNNENSLFLQMNDLRTEDTAMYYCARDMGWNFFQLQYWGQGTLVTVSS (SEQ ID NO: 103)CDR-H1 (DNA): GAC TTC ATC TTT AAA GAT TAT GCC (SEQ ID NO: 104)CDR-H1 (Polypeptide): D F I F K D Y A (SEQ ID NO: 105) CDR-H2 (DNA):ATT AGT GGT GAT GGT GAC ACT ACA (SEQ ID NO: 106) CDR-H2 (Polypeptide):I S G D G D T T (SEQ ID NO: 107) CDR-H3 (DNA):GCA AGA GAT ATG GGG TGG AAC TTC TTT CAG TTG CAA TAC (SEQ ID NO: 108)CDR-H3 (Polypeptide): A R D M G W N F F Q L Q Y (SEQ ID NO: 109)V_(L) domain (DNA):GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA (SEQ ID NO: 94) V_(L) domain (Polypeptide):DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (SEQ ID NO: 95) CDR-L1 (DNA):CAG AGC ATT AGC AGC TAT (SEQ ID NO: 96) CDR-L1 (Polypeptide):Q S I S S Y (SEQ ID NO: 97) CDR-L2 (DNA): GCT GCA TCC (SEQ ID NO: 98)CDR-L2 (Polypeptide): A A S (SEQ ID NO: 99) CDR-L3 (DNA):CAA CAG AGT TAC AGT ACC CCT CCG ATC ACC (SEQ ID NO: 100)CDR-L3 (Polypeptide): Q Q S Y S T P P I T (SEQ ID NO: 101)

See WO2017/218515.

The present invention further includes a complex comprising a C5polypeptide or an antigenic fragment thereof bound to one or moreanti-C5 antigen-binding proteins. For example, in an embodiment of theinvention, the complex comprises one or more C5 polypeptides orantigenic fragments thereof bound to one or more first anti-C5antigen-binding proteins and one or more further anti-C5 antigen-bindingproteins that do not compete for binding to the C5. Complexes can formwith various ratios of first antigen-binding protein to secondantigen-binding protein to C5. For example, the scope of the presentinvention includes a complex comprising:

(i) a 1:1:2, 2:2:4 or 3:3:6 ratio of first monospecific anti-C5antigen-binding protein (e.g., H4H12166P)-to-second monospecific anti-C5antigen-binding protein-to-C5 polypeptide or fragment(ii) a 1:1, 1:2, 2:1 or 2:2 ratio of bispecific anti-C5 antigen-bindingprotein-to-C5 polypeptide or fragment;(iii) a 1:1:1; 1:1:2 or 1:2:2 ratio of monospecific anti-C5antigen-binding protein-to-bispecific anti-C5 antigen-bindingprotein-to-C5 polypeptide or fragment; or(iv) a 1:2 ratio or monospecific anti-C5 antigen-binding protein-to-C5polypeptide or fragment.

In an embodiment of the invention, the monospecific anti-C5antigen-binding protein is eculizumab, H4H12166P, H4H12177P2 orH4H12176P2. In an embodiment of the invention, the bispecific anti-C5antigen-binding protein is H4H12176P2×H4H12177P2.

Some complexes were surmised based upon the calculated molar mass ofmaterial eluting after asymmetric flow field-flow fractionation(A4F-MALLS) and the average calculated masses of the individualantibodies and C5 polypeptide in the mixtures analyzed. These data areset forth in FIGS. 11-16 herein.

Epitope Mapping and Competition

As discussed herein, the present invention provides combinationsincluding a first antigen-binding protein (e.g., antibody orantigen-binding fragment thereof) that binds specifically to C5 and oneor more further antigen-binding proteins (e.g., antibodies orantigen-binding fragments thereof or polypeptides) (e.g., coversin) that(i) specifically bind to C5 at an epitope which is different from thatof the first antigen-binding protein and/or (ii) do not compete with thefirst antigen-binding protein for binding to C5.

Bispecific antigen-binding proteins (e.g., antibodies andantigen-binding fragments thereof) having two antigen-binding domains (afirst and a second) wherein the first specifically binds to an epitopeof C5 and the second (i) specifically bind to C5 at an epitope which isdifferent from that of the first antigen-binding domain and/or (ii) donot compete with the first antigen-binding domain for binding to C5.Such antigen-binding domains, in an embodiment of the invention, aretaken from the antibodies or antigen-binding fragments which are setforth in WO2017/218515.

Two antigen-binding proteins, e.g., antibodies, have a common epitope ifthere are common amino acids in the C5 antigen to which theantigen-binding proteins exhibit significant binding.

Methods for determining the epitope of an antigen-binding protein, e.g.,antibody or fragment or polypeptide, include alanine scanning mutationalanalysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide cleavage analysis, crystallographic studies and NMRanalysis. In addition, methods such as epitope excision, epitopeextraction and chemical modification of antigens can be employed (Tomer(2000) Prot. Sci. 9: 487-496). Another method that can be used toidentify the amino acids within a polypeptide with which anantigen-binding protein (e.g., antibody or fragment or polypeptide)(e.g., coversin) interacts is hydrogen/deuterium exchange detected bymass spectrometry. In general terms, the hydrogen/deuterium exchangemethod involves deuterium-labeling the protein of interest, followed bybinding the antigen-binding protein, e.g., antibody or fragment orpolypeptide, to the deuterium-labeled protein. Next, the C5protein/antigen-binding protein complex is transferred to water andexchangeable protons within amino acids that are protected by theantibody complex undergo deuterium-to-hydrogen back-exchange at a slowerrate than exchangeable protons within amino acids that are not part ofthe interface. As a result, amino acids that form part of theprotein/antigen-binding protein interface may retain deuterium andtherefore exhibit relatively higher mass compared to amino acids notincluded in the interface. After dissociation of the antigen-bindingprotein (e.g., antibody or fragment or polypeptide), the target proteinis subjected to protease cleavage and mass spectrometry analysis,thereby revealing the deuterium-labeled residues which correspond to thespecific amino acids with which the antigen-binding protein interacts.See, e.g., Ehring (1999) Analytical Biochemistry 267: 252-259; Engen andSmith (2001) Anal. Chem. 73: 256A-265A.

The term “epitope” refers to an antigenic determinant (e.g., on C5) thatinteracts with a specific antigen-binding site in an antigen-bindingprotein, e.g., variable region of an antibody molecule, known as aparatope. A single antigen may have more than one epitope. Thus,different antigen-binding proteins, e.g., antibodies, may bind todifferent areas on an antigen and may have different biological effects.Epitopes, composed of non-contiguous amino acids, may be referred to as“conformational”. A linear epitope contains only contiguous amino acids.In certain embodiments, epitopes may include determinants that arechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl groups, or sulfonyl groups, and, incertain embodiments, may have specific three-dimensional structuralcharacteristics, and/or specific charge characteristics.

For example, the epitope to which antibody H4H12166P bins is defined by:(i) the amino acid sequence NMATGMDSW which corresponds to amino acids591 to 599 in the beta chain included in SEQ ID NO: 1; and (ii) theamino acid sequence WEVHLVPRRKQLQFALPDSL, which corresponds to aminoacids 775 to 794 comprised in the alpha chain included in SEQ ID NO: 1.See for example, PCT International Application No. PCT/US2017/037226.The C5 epitope of Eculizumab is disclosed in Brachet et al., Eculizumabepitope on complement C5: Progress towards a better understanding of themechanism of action. Mol Immunol. 2016 September; 77: 126-131.

The term “competes” as used herein, refers to an antigen-binding protein(e.g., antibody or antigen-binding fragment thereof) that binds to anantigen and inhibits or blocks the binding of another antigen-bindingprotein (e.g., antibody or antigen-binding fragment thereof) to theantigen. The term also includes competition between two antigen-bindingprotein e.g., antibodies, in both orientations, i.e., a first antibodythat binds and blocks binding of second antibody and vice versa. Incertain embodiments, the first antigen-binding protein (e.g., antibody)and second antigen-binding protein (e.g., antibody) may bind to the sameepitope. Alternatively, the first and second antigen-binding proteins(e.g., antibodies) may bind to different, but, for example, overlappingepitopes, wherein binding of one inhibits or blocks the binding of thesecond antibody, e.g., via steric hindrance. Cross-competition betweenantigen-binding proteins (e.g., antibodies) may be measured by methodsknown in the art, for example, by a real-time, label-free bio-layerinterferometry assay. In an embodiment of the invention, competitionbetween a first and second anti-C5 antigen-binding protein (e.g.,antibody) is determined by measuring the ability of an immobilized firstanti-C5 antigen-binding protein (e.g., antibody) (not initiallycomplexed with C5 protein) to bind to soluble C5 protein complexed witha second anti-C5 antigen-binding protein (e.g., antibody). A reductionin the ability of the first anti-C5 antigen-binding protein (e.g.,antibody) to bind to the complexed C5 protein, relative to uncomplexedC5 protein, indicates that the first and second anti-C5 antigen-bindingproteins (e.g., antibodies) compete. The degree of competition can beexpressed as a percentage of the reduction in binding. Such competitioncan be measured using a real time, label-free bio-layer interferometryassay, e.g., on an Octet RED384 biosensor (Pall ForteBio Corp.), ELISA(enzyme-linked immunosorbent assays) or SPR (surface plasmon resonance).

Binding competition between anti-C5 antigen-binding proteins (e.g.,monoclonal antibodies (mAbs)) can be determined using a real time,label-free bio-layer interferometry assay on an Octet RED384 biosensor(Pall ForteBio Corp.). For example, to determine competition between twoanti-human C5 monoclonal antibodies, the anti-C5 mAb can be firstcaptured onto anti-hFc antibody coated Octet biosensor tips (PallForteBio Corp., #18-5060) by submerging the tips into a solution ofanti-human C5 mAb (subsequently referred to as “mAb1”). As apositive-control for blocking, the antibody captured biosensor tips canthen be saturated with a known blocking isotype control mAb(subsequently referred to as “blocking mAb”) by dipping into a solutionof blocking mAb. To determine if mAb2 competes with mAb1, the biosensortips can then be subsequently dipped into a co-complexed solution ofhuman C5 polypeptide and a second anti-human C5 mAb (subsequentlyreferred to as “mAb2”), that had been pre-incubated for a period of timeand binding of mAb1 to the C5 polypeptide can be determined. Thebiosensor tips can be washed in buffer in between every step of theexperiment. The real-time binding response can be monitored during thecourse of the experiment and the binding response at the end of everystep can be recorded. mAb2-dependent inhibition of mAb1/C5 bindingindicates competition between mAb1 and mAb2 for C5 binding. See e.g.,International Patent Application No. PCT/US2017/037226, filed Jun. 13,2017, e.g., Example 5 therein.

In an embodiment of the invention, competition between antigen-bindingproteins, such as antibodies, is determined under the conditions setforth in Example 5. For example, in an embodiment of the invention, theassay is conducted at 25° C. and pH about 7.4, e.g., in the presence ofbuffer (e.g., HEPES), salt (e.g., NaCl), surfactant (e.g., Tween-20) anda protein (e.g., bovine serum albumin), e.g., 0.01 M HEPES pH7.4, 0.15MNaCl, 0.05% v/v Surfactant Tween-20, 0.1 mg/mL BSA (Octet HBS-P buffer)with the plate shaking at the speed of 1000 rpm.

Competition between anti-C5 antibodies set forth in International PatentApplication No. PCT/US2017/037226 (WO2017/218515), filed Jun. 13, 2017is summarized below in Table 1. Accordingly, the present inventionincludes combinations comprising two anti-C5 antibodies orantigen-binding fragments thereof selected from Table 1 wherein theantibodies or fragments do not compete for C5 binding (e.g., H4H12166Pand H4H12168P; or H4H12166P and H4H12161P; or H4H12166P and H4H11686N).

TABLE 1 Competition between pairs of selected anti-C5 antibodies. FirstmAb (mAb1) Captured using mAb2 Antibodies Shown to Compete AHC Octetwith mAb1 H4H12183P2 H4H12167P; H4H12166P; H4H12163P H4H12167PH4H12183P2; H4H12166P; H4H12163P H4H12166P H4H12183P2; H4H12167P;H4H12163P H4H12163P H4H12183P2; H4H12167P; H4H12166P H4H12159PH4H12169P; H4H11683N; H4H12170P H4H12169P H4H12159P; H4H11683N;H4H12170P H4H11683N H4H12159P; H4H12169P; H4H12170P H4H12170P H4H12159P;H4H12169P; H4H11683N H4H12175P H4H12177P2 H4H12177P2 H4H12175PH4H12176P2 H4H12164P H4H12164P H4H12176P2 H4H12168P none H4H12161P noneH4H11686N none H4H12171P none

See WO2017/218515, Table 15. Pharmaceutical Compositions andAdministration

Combinations of the present invention (e.g., H4H12166P and one ofH4H12161P, H4H12170P, H4H12171P, H4H12175P, H4H12176P2 or H4H12177P2)include components that may be formulated into a single, commoncomposition or into multiple/separate compositions. Moreover, separatecompositions may be formulated with different varieties of carriers. Forexample, a first antigen-binding protein (e.g., antibody orantigen-binding fragment thereof) that binds specifically to C5, whichis part of a combination of the present invention, can be co-formulatedinto a single composition (e.g., with a pharmaceutically acceptablecarrier) with one or more further antigen-binding proteins (e.g.,antibodies or antigen-binding fragments thereof or polypeptides)(e.g.,coversin) that (i) specifically bind to C5 at an epitope which isdifferent from that of the first antigen-binding protein (e.g., antibodyor fragment) and/or (ii) do not compete with the first antigen-bindingprotein (e.g., antibody or fragment) for binding to C5. In embodiment ofthe invention, the first antigen-binding protein (e.g., antibody orfragment) and the second antigen-binding protein (e.g., antibody orfragment or polypeptide) are formulated into separate compositions(e.g., with pharmaceutically acceptable carriers). A further therapeuticagent, in a combination of the present invention, may be formulated intoyet another composition. A further therapeutic agent may be included ina combination of the present invention separately from the firstantibody or fragment and the second antibody or fragment or polypeptide.In another embodiment of the invention, the further therapeutic agent isformulated into either the first antibody or fragment or the secondantibody or fragment or polypeptide (or both).

To prepare pharmaceutical or sterile compositions comprising thecomponents of the combinations of the present invention, thecomponent(s) may be is admixed with a pharmaceutically acceptablecarrier or excipient. See, e.g., Remington's Pharmaceutical Sciences andU.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton,Pa. (1984). Combinations including such compositions are part of thepresent invention.

The scope of the present invention includes combinations including oneor more components in desiccated form, e.g., freeze-dried, substantiallylacking water.

Formulations may be prepared by mixing with acceptable carriers,excipients, or stabilizers in the form of, e.g., lyophilized powders,slurries, aqueous solutions or suspensions (see, e.g., Hardman, et al.(2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics,McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science andPractice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.;Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: ParenteralMedications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, etal. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, MarcelDekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety,Marcel Dekker, Inc., New York, N.Y.).

If a combination of the present invention includes a further therapeuticagent that is administered to a subject, the further therapeutic agentmay be is administered to the subject in accordance with the Physicians'Desk Reference (PDR), e.g., Physicians' Desk Reference 2003 (ThomsonHealthcare; 57th edition (Nov. 1, 2002)) and/or may formulated asdescribed in the PDR.

The mode of administration of a combination or any of the components ofa combination can vary. Routes of administration include oral, rectal,transmucosal, intestinal, parenteral; intramuscular, subcutaneous,intradermal, intramedullary, intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, intraocular, inhalation,insufflation, topical, cutaneous, transdermal, or intra-arterial.

The present invention provides methods for administering a combinationor component thereof comprising introducing the substance into the bodyof a subject. For example, the method comprises piercing the body of thesubject with a needle of a syringe and injecting the combination or acomponent thereof into the body of the subject, e.g., into the vein,artery, tumor, muscular tissue or subcutis of the subject.

The present invention provides a vessel (e.g., a plastic or glass vial,e.g., with a cap or a chromatography column, hollow bore needle or asyringe cylinder) comprising a combination of the present invention orone or more components thereof.

The present invention also provides an injection device comprising a oneor more antigen-binding proteins (e.g., antibody, antigen-bindingfragment or polypeptide) from a combination of the present invention ora pharmaceutical composition thereof. For example, one antigen-bindingprotein (from a combination) may be in a first injection device andanother antigen-binding protein (from the combination) may be in asecond injection device; or both antigen-binding proteins (from thecombination) may be in a common injection device. The injectiondevice(s) may be (co-)packaged into a kit. An injection device is adevice that introduces a substance into the body of a subject via aparenteral route, e.g., intramuscular, subcutaneous or intravenous. Forexample, an injection device may be a syringe (e.g., pre-filled with thepharmaceutical composition, such as an auto-injector) which, forexample, includes a cylinder or barrel for holding fluid to be injected(e.g., comprising the antibody or fragment or a pharmaceuticalcomposition thereof), a needle for piecing skin and/or blood vessels forinjection of the fluid; and a plunger for pushing the fluid out of thecylinder and through the needle bore. In an embodiment of the invention,an injection device that comprises an antigen-binding protein, e.g., anantibody or antigen-binding fragment thereof, from a combination of thepresent invention, or a pharmaceutical composition thereof is anintravenous (IV) injection device. Such a device can include theantigen-binding protein or a pharmaceutical composition thereof in acannula or trocar/needle which may be attached to a tube which may beattached to a bag or reservoir for holding fluid (e.g., saline)introduced into the body of the subject through the cannula ortrocar/needle. The antibody or fragment or a pharmaceutical compositionthereof may, in an embodiment of the invention, be introduced into thedevice once the trocar and cannula are inserted into the vein of asubject and the trocar is removed from the inserted cannula. The IVdevice may, for example, be inserted into a peripheral vein (e.g., inthe hand or arm); the superior vena cava or inferior vena cava, orwithin the right atrium of the heart (e.g., a central IV); or into asubclavian, internal jugular, or a femoral vein and, for example,advanced toward the heart until it reaches the superior vena cava orright atrium (e.g., a central venous line). In an embodiment of theinvention, an injection device is an autoinjector; a jet injector or anexternal infusion pump. A jet injector uses a high-pressure narrow jetof liquid which penetrate the epidermis to introduce the antibody orfragment or a pharmaceutical composition thereof to a subject's body.External infusion pumps are medical devices that deliver the antibody orfragment or a pharmaceutical composition thereof into a subject's bodyin controlled amounts. External infusion pumps may be poweredelectrically or mechanically. Different pumps operate in different ways,for example, a syringe pump holds fluid in the reservoir of a syringe,and a moveable piston controls fluid delivery, an elastomeric pump holdsfluid in a stretchable balloon reservoir, and pressure from the elasticwalls of the balloon drives fluid delivery. In a peristaltic pump, a setof rollers pinches down on a length of flexible tubing, pushing fluidforward. In a multi-channel pump, fluids can be delivered from multiplereservoirs at multiple rates.

The present invention includes methods for treating a C5-associateddisease or disorder (e.g., PNH or aHUS) in a subject (e.g., a human) inneed thereof by administering (e.g., parenterally), to the subject, atherapeutically effective amount of a combination comprising:

(1) a first antigen-binding protein (e.g., antibody or antigen-bindingfragment thereof) that binds specifically to C5 and one or more furtherantigen-binding proteins (e.g., polypeptides (e.g., coversin) orantibodies or antigen-binding fragments thereof) that (i) specificallybind to C5 at an epitope which is different from that of the firstantigen-binding protein (e.g., antibody or fragment) and/or (ii) doesnot compete with the first antigen-binding protein (e.g., antibody orfragment) for binding to C5; or(2) a multispecific antigen-binding protein (e.g., antibody orantigen-binding fragment thereof) (e.g., biparatopic anti-C5 IgGantibody) comprising two or more binding domains (e.g., first and secondbinding domain) that bind to different epitopes of C5 wherein the firstbinding domain (i) specifically binds to C5 at an epitope which isdifferent from that of the second binding domain and/or (ii) does notcompete with the second binding domain for binding to C5;optionally in association with a further therapeutic agent (e.g., acorticosteroid) and/or procedure (e.g., blood transfusion, e.g., in ahuman subject suffering from PNH).

“Treat” or “treating” means to administer a combination of the presentinvention, to a subject having one or more symptoms of a C5-associateddisease or disorder for which the combination is effective, e.g., in thetreatment of a subject having paroxysmal nocturnal hemoglobinuria (PNH)or atypical hemolytic uremic syndrome (aHUS), or being suspected ofhaving PNH or aHUS. Typically, the combination is administered in aneffective or therapeutically amount or dose (as discussed herein).

Guidance in selecting appropriate doses of combination of the present ora component thereof is available (see, e.g., Wawrzynczak (1996) AntibodyTherapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991)Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York,N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy inAutoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert et al. (2003)New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med.341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792;Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al.(2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J.Med. 343:1594-1602).

An effective or therapeutically effective dose of anti-C5antigen-binding protein (e.g., antibody or antigen-binding fragment orpolypeptide) in a combination of the invention for treating aC5-associated disease or disorder refers to the amount of thecombination sufficient to alleviate one or more signs and/or symptoms ofthe disease or disorder (e.g., an underlying cause such as complementactivation) in the treated subject or population, whether by inducingthe regression or elimination of such signs and/or symptoms or byinhibiting the progression of such signs and/or symptoms. The doseamount may vary depending upon the age and the size of a subject to beadministered, target disease, conditions, route of administration, andthe like. In an embodiment of the invention, an effective ortherapeutically effective dose of antigen-binding protein (e.g.,antibody or antigen-binding fragment thereof) of a combination of thepresent invention, for treating or preventing a C5-associated disease ordisorder, e.g., in an adult human subject, is a single dose of about 0.1to about 100 mg/kg body weight, e.g., about 5 to about 80, e.g., about10 to about 70, or about 20 to about 50 mg/kg body weight. Depending onthe severity of the condition, the frequency and the duration of thetreatment can be adjusted. In certain embodiments, the antigen-bindingprotein (e.g., antibody or antigen-binding fragment thereof) in acombination of the present invention can be administered as an initialdose of at least about 0.1 mg to about 800 mg, about 1 to about 600 mg,about 5 to about 500 mg, or about 10 to about 400 mg. In certainembodiments, the initial dose may be followed by administration of asecond or a plurality of subsequent doses of the antigen-binding protein(e.g., antibody or antigen-binding fragment thereof) in an amount thatcan be approximately the same or less than that of the initial dose,wherein the subsequent doses are separated by at least 1 day to 3 days;at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks;at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks;at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14weeks.

In an embodiment of the invention, coversin, in a combination of thepresent invention, is administered, for a first dose, at 0.57 mg/kg,followed by daily repeat maintenance doses wherein initial repeat doseis 25% of the ablating dose.

A “C5-associated” disease or disorder refers to a disease or disorderwhich is caused (directly or indirectly) by inflammation, cell injuryand/or cell killing that is mediated by C5a and/or C5b.

A C5-associated disease or disorder includes atypical hemolytic uremicsyndrome (aHUS). The present invention provides a method for treating orpreventing aHUS or for inducing the regression or elimination orinhibiting the progression of at least one sign or symptom of aHUS suchas:

-   -   platelet activation;    -   hemolysis;    -   systemic thrombotic microangiopathy (formation of blood clots in        small blood vessels throughout the body), e.g., leading to        stroke;    -   heart attack;    -   kidney failure (e.g., leading to death);    -   end-stage renal disease;    -   permanent renal damage;    -   abdominal pain;    -   confusion;    -   edema;    -   fatigue;    -   nausea/vomiting;    -   diarrhea; and/or    -   microangiopathic anemia,        in a subject in need thereof (e.g., in a subject suffering from        aHUS and suffering from one or more of such signs or symptoms)        by administering a therapeutically effective amount of the        combination to the subject.

A C5-associated disease or disorder includes paroxysmal nocturnalhemoglobinuria (PNH). The present invention provides a method fortreating or preventing PNH or for inducing the regression or eliminationor inhibiting the progression of at least one sign or symptom of PNHsuch as:

-   -   destruction of red blood cells;    -   thrombosis (e.g., deep vein thrombosis and/or pulmonary        embolism);    -   intravascular hemolytic anemia;    -   red discoloration of urine;    -   anemia;    -   tiredness;    -   shortness of breath;    -   heart palpitations;    -   abdominal pain; and/or    -   difficulty swallowing,        in a subject in need thereof (e.g., in a subject suffering from        aHUS and suffering from one or more of such signs or symptoms)        by administering a therapeutically effective amount of the        combination to the subject.

A C5-associated disease or disorder includes neurological disorders,renal disorders, multiple sclerosis, stroke, Guillain Barre Syndrome,traumatic brain injury, Parkinson's disease, disorders of inappropriateor undesirable complement activation, hemodialysis complications,hyperacute allograft rejection, xenograft rejection, interleukin-2induced toxicity during IL-2 therapy, inflammatory disorders,inflammation of autoimmune diseases, Crohn's disease, adult respiratorydistress syndrome, thermal injury including burns or frostbite,post-ischemic reperfusion conditions, myocardial infarction, capillaryleak syndrome, obesity, diabetes, Alzheimer's disease, schizophrenia,stroke, epilepsy, atherosclerosis, vasculitis, bullous pemphigoid, C3glomerulopathy, membraneproliferative glomerulonephritis, complementactivation caused by balloon angioplasty, post-pump syndrome incardiopulmonary bypass or renal bypass, complement activation caused byhemodialysis, renal ischemia, mesenteric artery reperfusion after aorticreconstruction, infectious disease or sepsis, immune complex disordersand autoimmune diseases, diabetic nephropathy, AIport's syndrome,progressive kidney failure, proteinuric kidney diseases, renalischemia-reperfusion injury, lupus nephritis, glomerulopathy, rheumatoidarthritis, systemic lupus erythematosus (SLE), SLE nephritis,membranoproliferative nephritis, hemolytic anemia, neuromyelitis optica,renal transplant, inherited CD59 deficiency, psoriasis, and myastheniagravis. The present invention includes methods for treating orpreventing any of the foregoing C5-related diseases or disorders, in asubject, by administering a therapeutically effective amount of acombination of the present invention to the subject in need thereof.

In certain other embodiments, the combinations of the present inventionare useful for treating or preventing at least one symptom or indicationof a C5-associated disease or disorder selected from the groupconsisting of lung disease and disorders such as dyspnea, hemoptysis,ARDS, asthma, chronic obstructive pulmonary disease (COPD), emphysema,pulmonary embolisms and infarcts, pneumonia, fibrogenic dust diseases,injury due to inert dusts and minerals (e.g., silicon, coal dust,beryllium, and asbestos), pulmonary fibrosis, organic dust diseases,chemical injury (due to irritant gasses and chemicals, e.g., chlorine,phosgene, sulfur dioxide, hydrogen sulfide, nitrogen dioxide, ammonia,and hydrochloric acid), smoke injury, thermal injury (e.g., burn,freeze), asthma, allergy, bronchoconstriction, hypersensitivitypneumonitis, parasitic diseases, Goodpasture's Syndrome, pulmonaryvasculitis, hereditary angioedema, and immune complex-associatedinflammation. The present invention includes methods for treating orpreventing any of the foregoing C5-related diseases or disorders, in asubject, by administering a therapeutically effective amount of acombination of the present invention to the subject in need thereof.

An ocular disease which is a C5-related disease or disorder includes,for example, age-related macular degeneration (AMD), diabetic macularedema (DME), diabetic retinopathy, ocular angiogenesis (ocularneovascularization affecting choroidal, corneal or retinal tissue),geographic atrophy (GA), uveitis and neuromyelitis optica. The presentinvention provides a method for treating or preventing an ocular diseaseor for inducing the regression or elimination or inhibiting theprogression of at least one sign or symptom of an ocular disease suchas:

-   -   increased rate of loss of vision;    -   drusen in the eye (e.g., of a subject with dry AMD);    -   loss of vision;    -   gradual loss of central vision (e.g., in subjects with        non-exudative macular degeneration);    -   visual distortion;    -   difficulty adapting to low light levels;    -   crooked central vision;    -   haziness of central and/or overall vision;    -   eye pigmentary changes;    -   distorted vision (e.g., metamorphopsia in which a grid of        straight lines appears wavy and parts of the grid may appear        blank);    -   exudative changes (e.g., hemorrhages in the eye, hard exudates,        subretinal/sub-RPE/intraretinal fluid);    -   slow recovery of visual function after exposure to bright light        (e.g., as determined in a photostress test);    -   incipient and/or geographic atrophy;    -   drastically decreasing visual acuity (e.g., two levels or more,        e.g., 20/20 to 20/80);    -   preferential hyperacuity perimetry changes (e.g., in a subject        with wet AMD);    -   blurred vision;    -   rapid onset of vision loss (e.g., caused by leakage and bleeding        of abnormal blood vessels in subjects with exudative macular        degeneration);    -   central scotomas (shadows or missing areas of vision);    -   trouble discerning colors (e.g., specifically dark colors from        other dark colors and/or light colors from other light colors);    -   loss in contrast sensitivity; and/or    -   straight lines appear curved in an Amsler grid,        in a subject in need thereof (e.g., in a subject suffering from        an ocular disease and suffering from one or more of such signs        or symptoms) by administering a therapeutically effective amount        of the combination to the subject.

It is also contemplated herein to administer a therapeutically effectiveamount of a combination of the present invention prophylactically tosubjects at risk for developing a C5-associated disease or disorder,e.g., aHUS, PNH or macular degeneration, such as subjects over the ageof 50, subjects with a family history of macular degeneration, smokers,and subjects with obesity, high cholesterol, cardiovascular disease,and/or unhealthy diet.

Combination Therapies

The present invention provides combinations comprising a firstantigen-binding protein (e.g., antibody or antigen-binding fragmentthereof) that binds specifically to C5 and one or more furtherantigen-binding proteins (e.g., polypeptides or antibodies orantigen-binding fragments thereof) that (i) specifically bind to C5 atan epitope which is different from that of the first antigen-bindingprotein (e.g., antibody or fragment) and/or (ii) does not compete withthe first antigen-binding protein (e.g., antibody or fragment) forbinding to C5 (e.g., H4H12166P and one of H4H12161P, H4H12170P,H4H12171P, H4H12175P, H4H12176P2 or H4H12177P2). Such combinations mayfurther include one or more further therapeutic agents and/or one ormore therapeutic methods. For example, the further therapeutic agent maybe formulated into a single composition with one or more components of acombination of the present invention or formulated separately from oneor both of the components. The present invention provides a method fortreating or preventing a C5-associated disease or disorder or fortreating or ameliorating at least one symptom or indication of such adisease or disorder in a subject in need thereof by administering atherapeutically effective amount of the combination to the subject,optionally in association with one or more further therapeutic agents.

In an embodiment of the invention, the further therapeutic agent isanother anti-C5 antibody or antigen-binding fragment thereof which isnot, itself a first or second/further antibody or fragment in thecombination, such as for example, one or more antibodies orantigen-binding fragments thereof selected from H2M11683N; H2M11686N;H4H12159P; H4H12163P; H4H12164P; H4H12166P2; H4H12166P3; H4H12166P4;H4H12166P5; H4H12166P6; H4H12166P7; H4H12166P8; H4H12166P9; H4H12166P10;H4H12167P; H4H12168P; H4H12169P; H4H12176P2; H4H12177P2; H4H12183P2;H2M11682N; H2M11684N; H2M11694N; and H2M11695N—as set forth inInternational PCT patent application no. PCT/US2017/037226 (or a variantthereof; or an antigen-binding protein such as an antibody orantigen-binding fragment that comprises a heavy chain immunoglobulinincluding CDR-H1, CDR-H2 and CDR-H3; and a light chain immunoglobulinincluding CDR-L1, CDR-L2 and CDR-L3 of any of the foregoing antibodies)(which is not a first or second/further antibody or antigen-bindingfragment in the combination).

Such a further therapeutic agent includes, for example, iron,antithymocyte globulin, a growth factor, an anti-coagulant (e.g.,warfarin, aspirin, heparin, phenindione, fondaparinux, idraparinux, andthrombin inhibitors such as argatroban, lepirudin, bivalirudin, ordabigatran) an anti-inflammatory drug (e.g., corticosteroids, andnon-steroidal anti-inflammatory drugs), an antihypertensive (e.g., anangiotensin converting enzyme inhibitor), an immunosuppressive agent(e.g., vincristine, cyclosporine A, or methotrexate), a fibrinolyticagent (e.g., ancrod, ε-aminocaproic acid, antiplasmin-a1, prostacyclin,and defibrotide), a lipid-lowering agent such as an inhibitor ofhydroxymethylglutaryl CoA reductase (e.g., atorvastatin), an anti-CD20agent such as rituximab, an anti-TNFα agent such as infliximab, ananti-seizure agent (e.g., magnesium sulfate), a C3 inhibitor, ananti-thrombotic agent, avacopan (CCX168; CAS#: 1346623-17-3),ravulizumab or zimura (avacincaptad pegol; CAS #1491144-00-3).

In an embodiment of the invention, the further therapeutic agent is anagent that inhibits an activity of C5; or C5 cleavage into C5a and C5b;or C5 expression. In an embodiment of the invention, the furthertherapeutic agent is C5 RNAi molecule or a polypeptide that binds to C5,e.g., a monoclonal antibody or peptide (e.g., cyclic peptide).

A further therapeutic agent that is administered to a subject inassociation with anti-C5 antibodies or antigen-binding fragments orpolypeptides are, in an embodiment of the invention, administered to thesubject in accordance with the Physicians' Desk Reference, e.g.,Physicians' Desk Reference 2003 (Thomson Healthcare; 57^(th) edition(Nov. 1, 2002)).

The further therapeutic agent may be administered to a subjectsequentially or simultaneously with administration of the components ofthe combinations of the invention. “Simultaneous” administration refersto the administration (e.g., injection) of two or more substances in asingle, common formulation or in separate formulations which areadministered during the same treatment session. “Sequential”administration refers to administration of two or more substances duringseparate treatment sessions (substantially separated by time). Forexample, a first component may be deemed to be administered, in ansequential administration regimen, “prior to” a second component e.g.,wherein the first component is administered 1 week before, 72 hoursbefore, 60 hours before, 48 hours before, 36 hours before, 24 hoursbefore, 12 hours before, 6 hours before, 5 hours before, 4 hours before,3 hours before, 2 hours before, 1 hour before, or 30 minutes beforeadministration of the second component. In other embodiments, theadditional therapeutically active component(s) may be administered to asubject after administration of an anti-C5 antibody of the presentinvention.

In an embodiment of the invention, the subject is further administered atherapeutic procedure, e.g., directed to the treatment of aC5-associated disease or disorder such as PNS or aHUS, e.g., dialysis, ablood or plasma transfusion or exchange and/or a bone marrow/stem celltransplant (BMT/SCT).

The present invention includes multispecific or multiparatopicantigen-binding proteins, as discussed herein, in association with afurther therapeutic agent, e.g., as discussed herein (e.g.,pharmaceutical compositions or kits thereof) as well as methods of usingsuch proteins to treat or prevent a C5-associated disease or disorder,e.g., as discussed herein.

Kits

The present invention provides kits comprising one or more components ofa combination of the present invention, optionally, in association withone or more further therapeutic agents, e.g., as discussed herein (e.g.,H4H12166P and one of H4H12161P, H4H12170P, H4H12171P, H4H12175P,H4H12176P2 or H4H12177P2). In one embodiment of the invention, the kitincludes an anti-C5 antigen-binding protein (e.g., antibody orantigen-binding fragment thereof) of the invention or a pharmaceuticalcomposition thereof in one device (e.g., pre-filled syringe) orcontainer (e.g., in a sterile glass or plastic vial) and another anti-C5antigen-binding protein (e.g., antibody or antigen-binding fragment) ofthe invention or a pharmaceutical composition thereof in another device(e.g., pre-filled syringe) or container (e.g., in a sterile glass orplastic vial).

In another embodiment, the kit comprises a combination of the invention,including two or more anti-C5 antigen-binding proteins (e.g., antibodiesor antigen-binding fragments) or a pharmaceutical composition thereof ina single, common container or device.

If the kit includes a pharmaceutical composition for parenteraladministration to a subject, the kit can include a device for performingsuch administration. For example, the kit can include one or morehypodermic needles or other injection devices as discussed above. Thus,the present invention includes a kit comprising an injection device andone or more anti-C5 antigen-binding protein (e.g., antibodies orantigen-binding fragments thereof) of the present invention, e.g.,wherein the injection device includes the antibody or fragment orwherein the antibody or fragment is in a separate vessel.

The kit can include a package insert including information concerningthe pharmaceutical compositions and dosage forms in the kit. Generally,such information aids patients/subjects and physicians in using theenclosed pharmaceutical compositions and dosage forms effectively andsafely. For example, the following information regarding a combinationof the invention may be supplied in the insert: pharmacokinetics,pharmacodynamics, clinical studies, efficacy parameters, indications andusage, contraindications, warnings, precautions, adverse reactions,overdosage, proper dosage and administration, how supplied, properstorage conditions, references, manufacturer/distributor information andpatent information.

The present invention includes methods for making a kit comprising acombination of the present invention. Such a method includes the stepsof co-packaging the first anti-C5 antigen-binding protein (e.g.,antibody or antigen-binding fragment); with the one or more of saidfurther antigen-binding proteins (e.g., polypeptides, antibodies orantigen-binding fragments) into a kit. The method optionally includesthe step of including one or more further therapeutic agents and/orother materials (e.g., as discussed herein) in the kit.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, room temperatureis about 25° C., and pressure is at or near atmospheric. Any antibodycombination or multispecific antibody set forth herein forms part of thepresent invention.

Example 1: Dual, but not Single, Anti C5 mAb Treatment Achieves CompleteInhibition of Alternative Complement Pathway Activation

The ability of various anti-C5 antibodies, individually or incombination with other agents and under various conditions, to inhibithemolysis was investigated.

Materials and Methods

Alternative Pathway Hemolysis Assay.

Alternative pathway hemolysis assay was used as the measure ofcomplement activation to evaluate the ability of anti-C5 mAbs to blockthe lysis of rabbit red blood cells (RbRBCs). Lysis of rabbit red bloodcells by membrane attack complex is the basis of the assay by whichcomplement activation is experimentally measured.

A desired number of RbRBCs are washed in GVB-Mg²⁺/EGTA buffer andresuspended at 2×10̂8 cells/ml. To test the efficacy of either singleanti-C5 mAb or a combination of anti-C5 mAbs, normal human serum wasdiluted to 50-96% in GVB-Mg²⁺/EGTA buffer to achieve a finalconcentration of 25-48% when added to RBC. Round bottom 96 well plateswere used to measure hemolysis activity. A total of 100 ul RbRBCs (2×10̂8cells/ml) were plated into 96-well plate at 37° C. followed by additionof 100 ul of diluted serum. Cells were gently mixed and incubated at 37°C. for 30-120 minutes. After incubation time, the cells were spun downby centrifugation at 1250×g at 4° C. A total of 100 uL of thesupernatant was transferred to a fresh 96 flat bottom plate and read at412 nm on a Spectramax microplate reader. The calculation of percent ofhemolysis was done as described below.

The percentage of hemolysis was calculated with the absorbance values byusing the following equation:

${\% \mspace{14mu} {Hemolysis}} = {100 \times \frac{\left( {{{Experimental}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}} - {{Background}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}}} \right)}{\left( {{{Maximum}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}} - {{Background}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}}} \right)}}$

In this equation “background cell lysis” was the OD at A412 nm from thecells incubated in GVB-Mg²⁺/EGTA buffer only containing no serum. The“maximum cell lysis” was the OD at A412 nm from the cells treated withwater. Maximum inhibition of lysis was calculated as a differencebetween bottom and top values in the curve expressed as a percentage oftop value. Data was represented as mean±Standard error of mean.

Anti-C5 Monoclonal Antibodies Tested.

A panel of 12 anti-C5 mAbs were tested, which included H4H12166P,H4H12170P, H4H12171P, H4H12175P, H4H12177P2, H4H12176P2, H4H12161P,H4H12183P2, H4H12159P, H4H12164P, H4H12167P, and H4H12163P. A Fabversion of H4H12170P was also evaluated in the assay.

Results

H4H12166P in Combination with Other Anti-C5 mAbs Completely BlockHemolysis of RbRBCs Via Alternative Pathway Activation.

As shown in FIG. 1A and Table 2A, under standard assay conditions of 25%NHS (normal human serum) and 30 min incubation time, all single mAbtreatments plateaued at around 80% inhibition (or less) of AP hemolysis.When used in a 1:1 molar ratio with H4H12166P, however, all combosblocked AP hemolysis down to essentially zero (FIG. 2B and Table 2B).

TABLE 2A Red blood cell lysis in the presence of single antibodies. %Antibody Max Inh. of Lysis H4H12166P 81.25 H4H12170P 88.66 H4H12161P59.24 H4H12171P 36.63 H4H12175P 77.83 H4H12176P2 62.80 H4H12177P2 76.21

TABLE 2B Red blood cell lysis in the presence of antibody combinations.% Antibody Combinations Max Inh. of Lysis H4H12166P + H4H12170P 97.59H4H12166P + H4H12161P 98.11 H4H12166P + H4H12171P 98.16 H4H12166P +H4H12175P 98.10 H4H12166P + H4H12176P2 97.29 H4H12166P + H4H12177P298.14

Inhibition with Combination Anti-C5 mAbs Persists at High SerumConcentrations or Longer Incubation Times.

Increasing incubation time from 30 min to 120 min (FIG. 2A and Table 3A)or serum concentration from 25% to 48% (120 minute incubation) (FIG. 2Band Table 3B) both significantly decreased the efficacy of inhibition bysingle mAbs. However, H4H12166P, in combination with H4H12161P, wasstill able to fully block AP hemolysis despite higher serumconcentrations or longer incubation times, demonstrating that blockadewas robust and complete.

TABLE 3A Red blood cell lysis in the presence of antibodies incubatedfor 30 or 120 minutes. % Max Inh. of Lysis Antibodies 30 min 120 minH4H12166P 82.79 57.21 H4H12161P 59.24 43.55 H4H12166P + 98.11 98.05H4H12161P

TABLE 3B Red blood cell lysis in the presence of antibodies and 25 or48% serum. % Max Inh. of Lysis 25% NHS 48% NHS Antibodies 120 min 120min H4H12166P 57.21 30.00 H4H12161P 43.55 17.37 H4H12166P + 98.05 96.26H4H12161P

Combo Effect is Also Observed with a Fab not Just mAbs, Also is notDependent on H4H12166P, but Requires the Combination mAbs from DifferentEpitope Bins.

A Fab version of H4H12170P when used in a 2:1 molar ratio withH4H12166P, also achieved complete blockade at both 30 min and 120 minincubation times (FIG. 3 and Table 4). Next, whether H4H12166P wasrequired for the observed combination effect was tested. As shown inFIG. 4 and Table 5, a different combination of antibodies, H4H12176P2and H4H12177P2, also offered complete blockade of RbRBC hemolysis viaalternative pathway showing the combination effect was independent ofH4H12166P. However, as shown in FIG. 5, the maximal inhibition ofhemolysis was not observed when testing combination mAbs from the sameepitope bins (H4H12170P+H4H12159P, H4H12175P+H4H12177P2,H4H12176P2+H4H12164P, H4H12167P+H4H12163P) demonstrating that differentbinding sites were required for this observed effect.

TABLE 4 Red blood cell lysis in the presence of a Fab version ofH4H12170P when used in a 2:1 molar ratio with H4H12166P. % Max Inh. ofLysis Antibodies 30 min 120 min H4H12166P 65.88 42.31 H4H12170P 76.4236.19 H4H12166P 8 + 98.14 98.10 H4H12170P

TABLE 5 Red blood cell lysis in the presence of H4H12176P2 and/orH4H12177P2 % Antibodies Max Inh. of Lysis H4H12176P2 61.88 H4H12177P275.12 H4H12176P2 + H4H12177P2 98.05

Addition of C3 LED to Decreased Blockade Effect of Single Anti-C5, butnot, Combination mAbs.

At 1 μM concentration with H4H12166P or H4H12161P mAbs, the addition ofsurplus human C3 protein resulted in partial recovery of AP activity inthe single but not combination mAb condition, suggesting that additionof C3 overcame the effect of single, but not, combination of anti-C5mAbs (FIG. 6).

Inhibition with Combination Anti-C5 mAbs does not Cause More Suppressionof C5a Generation Compared to Single Anti-C5 mAb.

As shown in FIG. 7, the blockade effects on C5a generation does notappear to be different between single (H4H12166P or H4H12161P) andcombination anti-C5 mAbs (H4H12166P+H4H12161P).

Example 2: A C5 Bispecific Antibody Achieves Complete Inhibition ofAlternative Complement Pathway Activation Similar to Combination ofAnti-C5 mAbs

Alternative pathway hemolysis assay was used as the measure ofcomplement activation to evaluate the ability of anti-C5 mAbs to blockthe lysis of rabbit red blood cells (RbRBCs). Lysis of rabbit red bloodcells by membrane attack complex is the basis of the assay by whichcomplement activation is experimentally measured.

A desired number of RbRBCs were washed in GVB-Mg²⁺/EGTA buffer andresuspended at 2×10⁸ cells/ml. To test the efficacy of either singleanti-C5 mAb or a combination of anti-C5 mAbs, normal human serum wasdiluted to 50-96% in GVB-Mg²⁺/EGTA buffer to achieve a finalconcentration of 25-48% when added to RBC. Round bottom 96 well plateswere used to measure hemolysis activity. A total of 100 ul RbRBCs (2×10⁸cells/ml) were plated into 96-well plate at 37° C. followed by additionof 100 ul of diluted serum. Cells were gently mixed and incubated at 37°C. for 30-120 minutes. After incubation time, the cells were spun downby centrifugation at 1250×g at 4° C. A total of 100 uL of thesupernatant was transferred to a fresh 96 flat bottom plate and read at412 nm on a Spectramax microplate reader. The calculation of percent ofhemolysis was done as described below.

The percentage of hemolysis was calculated with the absorbance values byusing the following equation:

${\% \mspace{14mu} {Hemolysis}} = {100 \times \frac{\left( {{{Experimental}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}} - {{Background}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}}} \right)}{\left( {{{Maximum}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}} - {{Background}\mspace{14mu} {Cell}\mspace{14mu} {Lysis}}} \right)}}$

In this equation, “background cell lysis” was the OD at A_(412nm) fromthe cells incubated in GVB-Mg²⁺/EGTA buffer only containing no serum.The “maximum cell lysis” was the OD at A_(412nm) from the cells treatedwith water. Maximum inhibition of lysis was calculated as a differencebetween bottom and top values in the curve expressed as a percentage oftop value. Data are represented as mean±Standard error of mean.

For the experiments to examine the molar ratio of mAb/C5, a fixedconcentration of 125 or 145 nM of C5 (purchased from CompTech Inc.) wasadded to the C5-deficient normal human serum (purchased from CompTechInc.) and titrated against various concentration of antibodies beforetesting in alternative pathway hemolysis assay.

Anti-C5 Monoclonal Antibodies Tested:

-   -   H412176P2    -   H412177P2    -   A bispecific antibody made from H412176P2 and H412177P2        (“H412176P2×H412177P2”)

H412176P2×H412177P2, a C5 Bispecific Antibody, Completely BlockedHemolysis of RbRBCs Via Alternative Pathway Activation.

As shown in FIG. 8 and Table 6, under standard assay conditions of 25%NHS and 30 min incubation times, a single mAb treatment of H412176P2 orH412177P2 led to partial inhibition of AP hemolysis. When used in a 1:1molar ratio, a combination of H412176P2+H412177P2, however, blocked APhemolysis down to essentially zero. H412176P2×H412177P2, a bispecificantibody made from the heavy and light Ig chains of H412176P2 andH412177P2, also showed complete suppression of AP hemolysis similar tothe combination of mAbs.

TABLE 6 Percentage of lysis inhibition % ABPID Max Inh. of LysisH4H12176P2 27.45 H4H12177P2 42.65 H4H12176P2 + H4H12177P2 98.10H412176P2xH412177P2 98.06

Near Molar Equivalent of Combination of Antibodies or C5-Bispecific isEnough to Completely Block Hemolysis of RbRBCs Via Alternative PathwayActivation.

As shown in FIG. 9, a mAb/C5 ratio of about 1.29 of either combinationmAbs (H412176P2+H412177P2) or C5 bispecific (H412176P2×H412177P2)blocked hemolysis to close to zero. Individual mAbs at this ratio, oreven above, offered only partial inhibition. Further experiments withH412176P2×H412177P2 showed a mAb/C5 ratio between 1.0-1.5 completelyblocked alternative pathway hemolysis assay (FIGS. 10A and 10B).

Example 3: Size Analysis of In Vitro Complexes Formed Between hC5 andAnti-hC5 Monoclonal Antibodies (mAbs) by Asymmetrical Flow Field-FlowFractionation Coupled to Multi-Angle Laser Light Scattering (A4F-MALLS)

The A4F-MALLS system is composed of an Eclipse™ 3+A4F Separation Systemcoupled to an Agilent 1200 Series HPLC system equipped with aultraviolet (UV) diode array detector, Wyatt Technology Dawn HELEOS® IIlaser light scattering instrument (LS), and an Optilab® T-rEXdifferential refractometer (RI) detector. The detectors were connectedin series in the following order: UV-LS-RI. LS and RI detectors werecalibrated according to instructions provided by Wyatt Technology.

Defined amounts of anti-hC5 mAbs were each combined with humancomplement C5 (hC5; EMD Millipore) and diluted in 1×DPBS, pH 7.4 toyield the final molar ratios listed in Table 7. All samples wereincubated at ambient temperature for 2 hours and maintained unfilteredat 4° C. prior to injection into an Eclipse™ short channel fitted with aW350 spacer foil (350 μm spacer thickness, 2.2 cm spacer width) andusing a 10 kDa MWCO Nadir regenerated cellulose membrane. The channelwas pre-equilibrated with the mobile phase buffer (10 mM sodiumphosphate, 500 mM sodium chloride, pH 7.0±0.1), prior to the injectionof each sample. Bovine serum albumin (BSA; 2 mg/mL; 10 μg sample load)was injected separately and included as a system suitability control.

The fractionation method consisted of four steps: injection, focusing,elution, and a channel “wash-out” step. The A4F-MALLS mobile phasebuffer (10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0±0.1) wasused throughout the fractionation method. Each sample (7 μg totalprotein load) was injected at a flow rate of 0.2 mL/min for 1 min andsubsequently focused for 2 min with a focus flow rate of 1.5 mL/min. Thesample was eluted with a channel flow rate of 1.0 mL/min and a lineargradient cross flow from 3.0 mL/min to 0 mL/min over 45 min. Finally,the cross flow was held at 0 mL/min for an additional 5 min to wash outthe channel. BSA was fractionated using the same parameter settings.

TABLE 7 Concentrations of Each Component for Sample PreparationmAb1:mAb2:hC5 Molar Ratio Sample (μM:μM:μM) Figure Table H4H12166P:hC51:1 11 9 H4H12166P:H4H12175P:hC5 0.5:0.5:1 12 10H4H12166P:H4H12177P2:hC5 0.5:0.5:1 12 10 H4H12166P:H4H12161P:hC50.5:0.5:1 13 10 H4H12166P:H4H12176P2:hC5 0.5:0.5:1 13 10H4H12176P2:H4H12177P2:hC5 0.5:0.5:1 13 10 H4H12166P:H4H12170P:hC50.5:0.5:1 14 10 H4H12166P:H4H12171P:hC5 0.5:0.5:1 15 10H4H12176P2xH4H12177P2 31 16 11 bispecific Ab:hC5 H4H12176P2xH4H12177P21:1 16 11 bispecific Ab:hC5 H4H12176P2xH4H12177P2 1:3 16 11 bispecificAb:hC5

A4F-MALLS Data Analysis.

Data were analyzed using ASTRA V software (version 5.3.4.14, WyattTechnology). The data were fit to the equation that relates the excessscattered light to the solute concentration and weight-average molarmass, Mw, (Kendrick B S, Kerwin B A, Chang B S, Philo J S. (2001). AnalBiochem. 299(2), 136-46, “Online Size-Exclusion High-Performance LiquidChromatography Light Scattering and Differential Refractometry Methodsto Determine Degree of Polymer Conjugation to Proteins andProtein-Protein or Protein-Ligand Association States”; Wyatt, P J.(1993) Anal. Chim. Acta 272(1), 1-40, “Light Scattering and the AbsoluteCharacterization of Macromolecules”):

$\begin{matrix}{\frac{K \star c}{R\left( {\theta,c} \right)} = {\frac{1}{{MwP}\; (\theta)} + {2A_{2}c}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

where c is the solute concentration, R(θ,c) is the excess Raleigh ratiofrom the solute as a function of scattering angle and concentration, Mwis the molar mass, P(θ) describes the angular dependence of scatteredlight (˜1 for particles with radius of gyration <50 nm), A₂ is thesecond virial coefficient in the expansion of osmotic pressure (whichcan be neglected since measurements are performed on dilute solutions)and K* is defined by Equation 2:

$\begin{matrix}{K \star {\frac{4\pi^{2}n_{0}^{2}}{N_{A}\lambda_{0}^{4}}\left( \frac{dn}{dc} \right)^{2}}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

where n_(o) represents the solvent refractive index, N_(A) is Avogadro'snumber, λ₀ is the wavelength of the incident light in a vacuum, anddn/dc represents the specific refractive index increment for the solute.

The normalization coefficients for the light scattering detectors,inter-detector delay volume and band broadening terms were calculatedfrom the BSA chromatograms collected for the A4F-MALLS conditionemployed. These values were applied to the data files collected for allother samples to correct for these terms.

The dn/dc value and the extinction coefficient at 215 nm (corrected forglycosylation) were experimentally determined using the proteinconjugate analysis provided in the Astra software. The correctedextinction coefficient and dn/dc value was used to analyze allprotein-protein complex samples. The molar mass of BSA monomer served toevaluate the calibration constants of the light scattering anddifferential refractive index detectors during data collection (systemsuitability check). The relative standard deviation of the average molarmass of BSA determined from the UV and RI detectors was 5.0%.

A4F-MALLS was used to assess the relative size distribution of complexesformed between anti-hC5 antibodies and hC5. The theoretical molar massof potential mAb:hC5 complexes along with their predicted stoichiometryis provided in Table 8.

Initial Screening of Anti-hC5 mAb Combinations Highlight Differences inSize Distribution of Complexes Formed with hC5.

In the absence of secondary mAbs, H4H12166P formed canonical 1:1 and 1:2complexes with hC5 when mixed in equimolar amounts (FIG. 11, Table 9).Overall, all mAb combinations examined exhibited the ability to formheteromeric complexes with hC5, with most combinations favoring asmaller, discrete species consistent with a 2:2 mAb:hC5 heteromericcomplex under the conditions tested (Peak 1, FIGS. 12 and 13; Table 10).Although minor amounts of larger, discrete complexes could also bedetected in these samples (Peak 2), formation of very large,heterogeneous, extended antibody-antigen lattices (≥˜1500 kDa; Peaks3-4)—a process termed “paper-dolling”—was limited. In contrast,combinations of H4H12166P with H4H12170P favored larger, moreheterogeneous complexes with hC5 indicative of a higher degree of“paper-dolling” compared to other combinations tested (FIG. 14; Table10). Finally, combinations of H4H12166P and H4H12171P displayed areduced tendency to form heteromeric complexes with hC5 as evidenced bythe presence of free mAb and 1:1 mAb:hC5 homomeric complex (*) detectedin this sample (FIG. 15; Table 10). This may suggest that the binding ofone mAb influences the affinity (and/or off-rate) of the other for hC5in this combination. Alternatively, this may indicate a reducedstability of the heteromeric complexes in this sample during thefractionation process compared to the other combinations tested.

Analysis of Complexes Formed Between H412176P2×H412177P2 (BispecificAnti-hC5 mAb) and hC5 Revealed a Stable 1:1 H412176P2×H412177P2:hC5Complex was Favored Under all Conditions.

When mixed at various molar ratios, H412176P2×H412177P2 bispecificantibody predominantly forms a stable 1:1 complex with hC5 suggestingthat both arms of the H412176P2×H412177P2 prefer to engage a singlemolecule of hC5—termed a monogamous, bivalent interaction (FIG. 16;Table 11). While minor amounts of additional discrete complexesconsistent with 1:2, 2:1, and 2:2 mAb:hC5 could be detected undervarious conditions, no additional higher order complexes were observedindicating that H412176P2×H412177P2 does not promote “paper-dolling”with hC5.

TABLE 8 Theoretical Molar Mass of mAb:hC5 Complexes mAb:hC5 ComplexTheoretical Molar Mass (kDa) 1:0 150 0:1 195 1:1 345 2:1 495 1:2 540 2:2690 3:2 840 2:3 885 3:4 1230 4:4 1380 5:5 1725 6:5 1875 5:6 1920 6:62070

TABLE 9 Summary Table of Approximate Molar Mass and Retention Time ofhC5 Complexes with H4H12166P Alone Peak 1 Peak 2 Molar [mAb]₁:[hC5]₁[mAb]₁:[hC5]₂ Ratio Complex Complex Sample (μM:μM) R_(t), min M_(w), kDaR_(t), min M_(w), kDa H4H12166P:hC5 1:1 13.7 341.1 15.3 498.7 R_(t):Retention Time; M_(w): weight average molar mass; min: minutes; kDa:kiloDaltons;

TABLE 10 Summary Table of Approximate Molar Mass and Retention Time ofhC5 Complexes with Anti-hC5 mAb Combinations Peak 4 Higher Order Peak 1Peak 2 Peak 3 Heteromeric Molar [mAb]₁₋₂:hC5]₂ [mAb]₃₋₄:[hC5]₄[mAb]₅₋₆:[hC5]₅₋₆ Complexes Ratio Complex Complex Complex([mAb]_(≥7):[hC5]_(≥7)) Sample (μM:μM:μM) R_(t), min M_(w), kDa R_(t),min M_(w), kDa R_(t), min M_(w), kDa R_(t), min M_(w), kDaH4H12166P:H4H12175P:hC5 0.5:0.5:1 16.0 684.4 18.5 1342.4 20.1 1876.021.5 ~2250-3560 H4H12166P:H4H12177P2:hC5 0.5:0.5:1 16.1 687.7 18.51327.4 20.1 1865.6 21.5 ~2380-4250 H4H12166P:H4H12161P:hC5 0.5:0.5:116.4 684.7 18.4 1261.8 NA NA 20.2 ~1700-2700 H4H12166P:H4H12176P2:hC50.5:0.5:1 15.7 685.9 17.7 1319.8 19.4 1849.8 20.6 ~2300-3800H4H12176P2:H4H12177P2:hC5 0.5:0.5:1 15.8 687.7 17.8 1333.8 19.3 1871.620.6 ~2300-3600 H4H12166P:H4H12170P:hC5 0.5:0.5:1 15.5 664.8 19.2 1304.321.9 1901.1 23.6 ~2300-4100 H4H12166P:H4H12171P:hC5 0.5:0.5:1 15.9 649.619.1 1288.2 ND ND 20.6 ~1700-2300 R_(t): Retention Time; M_(w): weightaverage molar mass; min: minutes; kDa: kiloDaltons; ND: not detected

TABLE 11 Summary Table of Approximate Molar Mass and Retention Time ofhC5 Complexes with H412176P2 × H412177P2 (Anti-hC5 Bispecific mAb) Peak1 Peak 2 Peak 3 Peak 4 Molar [mAb]₁:[hC5]₁ [mAb]₂:[hC5]₁ [mAb]₁:[hC5]₂[mAb]₂:[hC5]₂ Ratio Complex Complex Complex Complex Sample (μM:μM)R_(t), min M_(w), kDa R_(t), min M_(w), kDa R_(t), min M_(w), kDa R_(t),min M_(w), kDa H412176P2 × H412177P2:hC5 3:1 11.2 359.3 11.9 506.5 N/AN/A N/A N/A H412176P2 × H412177P2:hC5 1:1 11.2 347.2 ND ND N/A N/A 12.3681.3 H412176P2 × H412177P2:hC5 1:3 11.1 350.9 N/A N/A 12.2 559.5 ND NDR_(t): Retention Time; M_(w): weight average molar mass; min: minutes;kDa: kiloDaltons; ND: not detected

1. A combination comprising a first antigen-binding protein that bindsspecifically to C5; and one or more further antigen-binding proteinsthat (i) specifically bind to C5 at an epitope which is different fromthat of the first antigen-binding protein; and/or (ii) does not competewith the first antigen-binding protein for binding to C5.
 2. Thecombination of claim 1 wherein the first antigen-binding protein is anantibody or antigen-binding fragment that specifically binds to C5; andthe further antigen-binding protein is an antibody or antigen-bindingfragment or polypeptide that specifically binds to C5.
 3. Thecombination of claim 1, comprising a first antibody or antigen-bindingfragment thereof which is H4H12166P; and a further antibody orantigen-binding fragment thereof which is one or more selected from thegroup consisting of: H2M11683N; H2M11686N; H4H12159P; H4H12161P;H4H12164P; H4H12166P2; H4H12166P3; H4H12166P4; H4H12166P5; H4H12166P6;H4H12166P7; H4H12166P8; H4H12166P9; H4H12166P10; H4H12168P; H4H12169P;H4H12170P; H4H12171P; H4H12175P; H4H12176P2; H4H12177P2; H2M11682N;H2M11684N; H2M11694N and H2M11695N.
 4. The combination of claim 1,wherein the first antigen-binding protein comprises: CDR-H1, CDR-H2 andCDR-H3 of a heavy chain variable region of antibody H4H12166P; andCDR-L1, CDR-L2 and CDR-L3 of a light chain variable region of antibodyH4H12166P; and the further antigen-binding protein comprises: (i)CDR-H1, CDR-H2 and CDR-H3 of a heavy chain variable region of antibodyH4H12161P; and CDR-L1, CDR-L2 and CDR-L3 of a light chain variableregion of antibody H4H12161P; (ii) CDR-H1, CDR-H2 and CDR-H3 of a heavychain variable region of antibody H4H12170P; and CDR-L1, CDR-L2 andCDR-L3 of a light chain variable region of antibody H4H12170P; (iii)CDR-H1, CDR-H2 and CDR-H3 of a heavy chain variable region of antibodyH4H12171P; and CDR-L1, CDR-L2 and CDR-L3 of a light chain variableregion of antibody H4H12171P; (iv) CDR-H1, CDR-H2 and CDR-H3 of a heavychain variable region of antibody H4H12175P; and CDR-L1, CDR-L2 andCDR-L3 of a light chain variable region of antibody H4H12175P; (v)CDR-H1, CDR-H2 and CDR-H3 of a heavy chain variable region of antibodyH4H12176P2; and CDR-L1, CDR-L2 and CDR-L3 of a light chain variableregion of antibody H4H12176P2; or (vi) CDR-H1, CDR-H2 and CDR-H3 of aheavy chain variable region of antibody H4H12177P2; and CDR-L1, CDR-L2and CDR-L3 of a light chain variable region of antibody H4H12177P2. 5.The combination of claim 1, wherein the first antigen-binding protein isantibody H4H12166P or an antibody or antigen-binding fragment thereofcomprising the V_(H) and V_(L) thereof; and the further antigen-bindingprotein is antibody H4H12161P, H4H12170P, H4H12171P, H4H12175P,H4H12176P2 or H4H12177P2; or an antibody or antigen-binding fragmentthereof comprising the V_(H) and V_(L) thereof.
 6. The combination ofclaim 1, wherein the first antigen-binding protein and the furtherantigen-binding protein are formulated in a single composition.
 7. Thecombination of claim 1 wherein the further antigen-binding protein is apolypeptide which is coversin.
 8. The combination of claim 1 wherein thefurther antigen-binding protein is an antibody which is eculizumab. 9.The combination of claim 1, which comprises a further therapeutic agent.10. The combination of claim 1, which comprises a further therapeuticagent which is an antibody or antigen-binding fragment that specificallybinds to C5.
 11. The combination of claim 1, which comprises a furthertherapeutic agent which is an antibody that specifically binds to C5which is selected from the group consisting of: H2M11683N; H2M11686N;H4H12159P; H4H12163P; H4H12164P; H4H12166P2; H4H12166P3; H4H12166P4;H4H12166P5; H4H12166P6; H4H12166P7; H4H12166P8; H4H12166P9; H4H12166P10;H4H12167P; H4H12168P; H4H12169P; H4H12176P2; H4H12177P2; H4H12183P2;H2M11682N; H2M11684N; H2M11694N; and H2M11695N; or an antigen-bindingfragment thereof; or which is an antibody that binds to C5, ananti-coagulant, a thrombin inhibitor, an anti-inflammatory drug, anantihypertensive, an immunosuppressive agent, a fibrinolytic agent, alipid-lowering agent, an inhibitor of hydroxymethylglutaryl CoAreductase, an anti-CD20 agent, an anti-TNFα agent, an anti-seizureagent, a C3 inhibitor or an anti-thrombotic agent.
 12. The combinationof claim 1, which comprises a further therapeutic agent selected fromthe group consisting of: warfarin, aspirin, heparin, phenindione,fondaparinux, idraparinux, argatroban, lepirudin, bivalirudin, ordabigatran, corticosteroids, and non-steroidal anti-inflammatory drugs,vincristine, cyclosporine A, methotrexate, ancrod, ε-aminocaproic acid,antiplasmin-a1, prostacyclin, defibrotide, rituximab and magnesiumsulfate.
 13. A bispecific or biparatopic antibody or antigen-bindingfragment thereof comprising a first antigen-binding domain thatspecifically binds to C5 at a first epitope and a second antigen-bindingdomain that (i) specifically binds to C5 at a second epitope which isdifferent from that of the first antigen-binding domain and/or (ii) doesnot compete with the first antigen-binding domain for binding to C5. 14.The bispecific or biparatopic antibody or fragment of claim 13 which isan IgG.
 15. A bispecific or biparatopic antigen-binding protein selectedfrom the group consisting of: H4H12161P×H4H12177P2;H4H12166P×H4H12177P2; H4H12170P×H4H12177P2; H4H12171P×H4H12177P2;H4H12175P×H4H12177P2; H4H12176P2×H4H12177P2; H4H12176P2×H4H12161P;H4H12176P2×H4H12166P; H4H12176P2×H4H12170P; H4H12176P2×H4H12171P;H4H12176P2×H4H12175P; H4H12175P×H4H12161P; H4H12175P×H4H12166P;H4H12175P×H4H12170P; H4H12175P×H4H12171P; H4H12171P×H4H12161P;H4H12171P×H4H12166P; H4H12171P×H4H12170P; H4H12170P×H4H12161P;H4H12170P×H4H12166P; and H4H12166P×H4H12161P.
 16. A complex comprising:one or more C5 polypeptides or antigenic fragments thereof bound to oneor more first anti-C5 antigen-binding proteins and one or more furtheranti-C5 antigen-binding proteins that do not compete for binding to theC5.
 17. A complex comprising: (i) a 1:1:2, 2:2:4 or 3:3:6 ratio of firstmonospecific anti-C5 antigen-binding protein-to-second monospecificanti-C5 antigen-binding protein-to-C5 polypeptide or antigenic fragmentthereof; (ii) a 1:1, 1:2, 2:1 or 2:2 ratio of bispecific anti-C5antigen-binding protein-to-C5 polypeptide or antigenic fragment thereof;or (iii) a 1:1:1; 1:1:2 or 1:2:2 ratio of ratio of monospecific anti-C5antigen-binding protein-to-bispecific anti-C5 antigen-bindingprotein-to-C5 polypeptide or antigenic fragment thereof.
 18. A methodfor treating or preventing a C5-associated disease or disorder in asubject and/or for inhibiting both the classical complement pathway andthe alternative complement pathway in a subject in need of suchtreatment, prevention and/or inhibition, the method comprisingadministering, to the subject, a first antigen-binding protein thatspecifically binds C5 and a second antigen-binding protein thatspecifically binds C5; wherein the first and second antigen-bindingproteins: (a) bind to distinct, non-overlapping epitopes on C5; and/or(b) do not compete with one another for binding to C5 and/or amultispecific antigen-binding protein that specifically binds C5;wherein the multispecific antigen-binding protein comprises a first andsecond antigen-binding domain wherein the domains (a) bind to distinct,non-overlapping epitopes on C5; and/or (b) do not compete with oneanother for binding to C5.
 19. A method for treating or preventing aC5-associated disease or disorder in a subject in need of such treatmentor prevention comprising administering an effective amount of thecombination of claim
 1. 20. The method of claim 18, wherein theC5-associated disease or disorder is selected from the group consistingof: Acute respiratory distress syndrome; adult respiratory distresssyndrome; age-related macular degeneration; allergy; AIport's syndrome;Alzheimer's disease; asthma; asthma; atherosclerosis; atypical hemolyticuremic syndrome; autoimmune diseases; complement activation caused byballoon angioplasty; bronchoconstriction; bullous pemphigoid; burns; C3glomerulopathy; capillary leak syndrome; chemical injury; chronicobstructive pulmonary disease; Crohn's disease; diabetes; diabeticmacular edema; diabetic nephropathy; diabetic retinopathy; dyspnea;emphysema; epilepsy; fibrogenic dust diseases; frostbite; geographicatrophy; glomerulopathy; Goodpasture's Syndrome; Guillain-BarreSyndrome; complement activation caused by hemodialysis; hemodialysiscomplications; hemolytic anemia; hemoptysis; hereditary angioedema;hyperacute allograft rejection; hypersensitivity pneumonitis; immunecomplex disorders; immune complex-associated inflammation; inflammationof autoimmune diseases; inflammatory disorders; inherited CD59deficiency; injury due to inert dusts and/or minerals; interleukin-2induced toxicity during IL-2 therapy; lupus nephritis;membraneproliferative glomerulonephritis; membranoproliferativenephritis; mesenteric artery reperfusion after aortic reconstruction;mesenteric artery reperfusion after infectious disease; mesentericartery reperfusion after sepsis; multiple sclerosis; myasthenia gravis;myocardial infarction; neuromyelitis optica; neuromyelitis optica;obesity; ocular angiogenesis; organic dust diseases; parasitic diseases;Parkinson's disease; paroxysmal nocturnal hemoglobinuria; pneumonia;post-ischemic reperfusion conditions; post-pump syndrome incardiopulmonary bypass or renal bypass; progressive kidney failure;proteinuric kidney diseases; psoriasis; pulmonary embolisms andinfarcts; pulmonary fibrosis; pulmonary vasculitis; renal ischemia;renal ischemia-reperfusion injury; renal transplant; rheumatoidarthritis; schizophrenia; smoke injury; stroke; stroke; systemic lupuserythematosus; systemic lupus erythematosus nephritis; thermal injury;thermal injury; traumatic brain injury; uveitis; vasculitis; andxenograft rejection.
 21. The method of claim 18, wherein the subject isadministered one or more further therapeutic agents and/or one or moretherapeutic procedures.
 22. The method of claim 21 wherein the subjectis administered a further therapeutic agent which is an antibody orantigen-binding fragment that specifically binds to C5.
 23. The methodclaim of 21 wherein the subject is administered one or more furthertherapeutic agents which is an antibody that binds to C5 which isselected from the group consisting of: H2M11683N; H2M11686N; H4H12159P;H4H12163P; H4H12164P; H4H12166P2; H4H12166P3; H4H12166P4; H4H12166P5;H4H12166P6; H4H12166P7; H4H12166P8; H4H12166P9; H4H12166P10; H4H12167P;H4H12168P; H4H12169P; H4H12176P2; H4H12177P2; H4H12183P2; H2M11682N;H2M11684N; H2M11694N; and H2M11695N; or an antigen-binding fragmentthereof; or which is selected from the group consisting of: an antibodythat binds to C5, an anti-coagulant, a thrombin inhibitor, ananti-inflammatory drug, an antihypertensive, an immunosuppressive agent,a fibrinolytic agent, a lipid-lowering agent, an inhibitor ofhydroxymethylglutaryl CoA reductase, an anti-CD20 agent, an anti-TNFαagent, an anti-seizure agent, a C3 inhibitor and an anti-thromboticagent; and/or wherein the subject is administered a therapeuticprocedure which is dialysis, a blood or plasma transfusion or exchangeand/or a bone marrow/stem cell transplant (BMT/SCT).
 24. The methodclaim 21 wherein the subject is administered one or more furthertherapeutic agents selected from the group consisting of: eculizumab,coversin, iron, antithymocyte globulin, a growth factor, warfarin,aspirin, heparin, phenindione, fondaparinux, idraparinux, argatroban,lepirudin, bivalirudin, or dabigatran, corticosteroids, andnon-steroidal anti-inflammatory drugs, vincristine, cyclosporine A,methotrexate, ancrod, ε-aminocaproic acid, antiplasmin-a1, prostacyclin,defibrotide, rituximab, magnesium sulfate, avacopan, ravulizumab andavacincaptad pegol.
 25. The method of claim 18, wherein one or more ofthe components of the combination are administered to the subjectsubcutaneously, intravenously, intradermally, intraperitoneally, orally,intramuscularly or intracranially.
 26. The method of claim 18, whereinthe subject is human.
 27. A method for making the combination of claim1, comprising co-packaging: said first antigen-binding protein; said oneor more of further antigen-binding protein; and, optionally, one or morefurther therapeutic agents, into a kit.
 28. A method for making thecombination of claim 1, comprising co-formulating: said firstantigen-binding protein, said one or more further antigen-bindingproteins; and optionally, one or more further therapeutic agents; and apharmaceutically acceptable carrier into a single pharmaceuticalformulation and, optionally, incorporating the formulation into a deviceor vessel.
 29. A combination which is the product of the method of claim27.